Maize cellulose synthases and uses thereof

ABSTRACT

The invention provides isolated cellulose synthase nucleic acids and their encoded proteins. The present invention provides methods and compositions relating to altering cellulose synthase levels in plants. The invention further provides recombinant expression cassettes, host cells, and transgenic plants comprising said nucleic acids.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/486,129 filed Jun. 17, 2009, which is a divisional of U.S. patentapplication Ser. No. 12/277,418 filed Nov. 25, 2008, now issued as U.S.Pat. No. 7,579,443, which is a divisional of U.S. patent applicationSer. No. 11/859,968 filed Sep. 24, 2007, now issued as U.S. Pat. No.7,524,933, which is a divisional of U.S. patent application Ser. No.10/963,217 filed Oct. 12, 2004, now issued as U.S. Pat. No. 7,307,149,which is a continuation-in-part of U.S. patent application Ser. No.10/209,059, filed Jul. 31, 2002, now issued as U.S. Pat. No. 6,930,225,which is a continuation-in-part of U.S. patent application Ser. No.09/550,483, filed Apr. 14, 2000, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 09/371,383,filed Aug. 6, 1999, now abandoned, which claims benefit of U.S.Provisional Patent Application Ser. No. 60/096,822, filed Aug. 17, 1998,now abandoned, all of which are incorporated herein by reference. Alsoincorporated by reference are U.S. patent application Ser. No.11/493,187, filed Jul. 26, 2006, now issued as U.S. Pat. No. 7,312,377and U.S. patent application Ser. No. 10/961,254 filed Oct. 8, 2004, nowissued as U.S. Pat. No. 7,214,852, U.S. patent application Ser. No.10/160,719, filed Jun. 3, 2002, now issued as U.S. Pat. No. 6,803,498,which is a continuation of U.S. patent application Ser. No. 09/371,383,filed Aug. 6, 1999, now abandoned, which claims benefit of U.S.Provisional Patent Application Ser. No. 60/096,822, filed Aug. 17, 1998,now abandoned.

TECHNICAL FIELD

The present invention relates generally to plant molecular biology. Morespecifically, it relates to nucleic acids and methods for modulatingtheir expression in plants.

BACKGROUND OF THE INVENTION

Polysaccharides constitute the bulk of the plant cell walls and havebeen traditionally classified into three categories: cellulose,hemicellulose and pectin. Fry, (1988) The growing plant cell wall:Chemical and metabolic analysis, New York: Longman Scientific &Technical. Whereas cellulose is made at the plasma membrane and directlylaid down into the cell wall, hemicellulosic and pectic polymers arefirst made in the Golgi apparatus and then exported to the cell wall byexocytosis. Ray, et al., (1976) Ber. Deutsch. Bot. Ges. Bd. 89:121-146.The variety of chemical linkages in the pectic and hemicellulosicpolysaccharides indicates that there must be tens of polysaccharidesynthases in the Golgi apparatus. Darvill, et al., (1980) The primarycell walls of flowering plants. In The Plant Cell (N. E. Tolbert, ed.),Vol. 1 in Series: The biochemistry of plants: A comprehensive treatise,eds. Stumpf and Conn, (New York: Academic Press), pp. 91-162.

Even though sugar and polysaccharide compositions of the plant cellwalls have been well characterized, very limited progress has been madetoward identification of the enzymes involved in polysaccharidesformation, the reason being their labile nature and recalcitrance tosolubilization by available detergents. Sporadic claims for theidentification of cellulose synthase from plant sources were made overthe years. Callaghan and Benziman, (1984) Nature 311:165-167; Okuda, etal., (1993) Plant Physiol. 101:1131-1142. However, these claims were metwith skepticism. Callaghan and Benziman, (1985), Nature 314:383-384;Delmer, et al., (1993) Plant Physiol. 103:307-308. It was onlyrelatively recently that a putative gene for plant cellulose synthase(CesA) was cloned from the developing cotton fibers based on homology tothe bacterial gene. Pear, et al., Proc. Natl. Acad. Sci. USA93:12637-12642; Saxena, et al., (1990) Plant Molecular Biology15:673-684; see also, WO 98/18949; see also, Arioli, et al., (1998).Molecular analysis of cellulose biosynthesis in Arabidopsis. ScienceWashington DC. Jan. 279:717-720. A number of genes for cellulosesynthase family were later isolated from other plant species based onsequence homology to the cotton gene (Richmond and Somerville, (2000)Plant Physiology 124:495-498.)

Cellulose, by virtue of its ability to form semicrystallinemicrofibrils, has a very high tensile strength which approaches that ofsome metals. Niklas, (1992), Plant Biomechanics: An engineering approachto plant form and function, The University of Chicago Press, p. 607.Bending strength of the culm of normal and brittle-culm mutants ofbarley has been found to be directly correlated with the concentrationof cellulose in the cell wall. Kokubo, et al., (1989), Plant Physiology91:876-882; Kokubo, et al., (1991) Plant Physiology 97:509-514.

Although stalk composition contributes to numerous quality factorsimportant in maize breeding, little is known in the art about the impactof cellulose levels on such agronomically important traits as stalklodging, silage digestibility or downstream processing. The presentinvention provides these and other advantages.

SUMMARY OF THE INVENTION

Generally, it is the object of the present invention to provide nucleicacids and proteins relating to cellulose synthases. It is an object ofthe present invention to provide transgenic plants comprising thenucleic acids of the present invention and methods for modulating, in atransgenic plant, expression of the nucleic acids of the presentinvention.

Therefore, in one aspect the present invention relates to an isolatednucleic acid comprising a member selected from the group consisting of(a) a polynucleotide having a specified sequence identity to apolynucleotide encoding a polypeptide of the present invention; (b) apolynucleotide which is complementary to the polynucleotide of (a) and(c) a polynucleotide comprising a specified number of contiguousnucleotides from a polynucleotide of (a) or (b). The isolated nucleicacid can be DNA.

In other aspects the present invention relates to: 1) recombinantexpression cassettes, comprising a nucleic acid of the present inventionoperably linked to a promoter, 2) a host cell into which has beenintroduced the recombinant expression cassette, 3) a transgenic plantcomprising the recombinant expression cassette and 4) a transgenic plantcomprising a recombinant expression cassette containing more than onenucleic acid of the present invention each operably linked to apromoter. Furthermore, the present invention also relates to combiningby crossing and hybridization recombinant cassettes from differenttransformants. The host cell and plant are optionally from maize, wheat,rice or soybean.

In other aspects the present invention relates to methods of alteringstalk lodging and other standability traits, including, but not limitedto brittle snap and improving stalk digestibility, through theintroduction of one or more of the polynucleotides that encode thepolypeptides of the present invention. Additional aspects of the presentinvention include methods and transgenic plants useful in the end useprocessing of compounds such ads cellulose or use of transgenic plantsas end products either directly, such as silage, or indirectly followingprocessing, for such uses known to those of skill in the art, such as,but not limited to, ethanol. Also, one of skill in the art wouldrecognize that the polynucleotides and encoded polypeptides of thepresent invention can be introduced into an host cell or transgenicplant wither singly or in multiples, sometimes referred to in the art as“stacking” of sequences or traits. It is intended that thesecompositions and methods be encompassed in the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Stalk breaking strength of hybrids and its comparison with thelodging scores. The mechanical strength is very similar to the lodgingscores that have been assigned based on field observations. The verticallight-colored bar in the upper right corner of the figure is the leastsignificant difference (LSD) estimate at 5% level.

FIG. 2: Stalk strength of T_(o) transgenic plants. Plants overexpressing(“up”) ZmCesA4 and ZmCesA8 had significantly stronger stalks than thecontrols. Overexpression of CesA5 did not alter stalk strength whereasthe overexpression of CesA1 led to weaker and stunted stalks.

FIG. 3: Correlation between unit cellulose and stalk breaking strength.Stalk breaking strength was highly correlated with the amount ofcellulose in a unit stalk length. (correlation coefficient, r: 0.76;0.63; 0.92; 0.86.) While these correlations are specifically related tothe different CesA genes, it should be noted that in general the samecorrelation would apply. In other words, it is expected this would applyto low cellulose levels as well as higher cellulose levels.

FIG. 4: Unrooted cladogram of CesA proteins from different species.Sequences are labeled by prefixes. This cladogram demonstrates therelevance of the maize genes to those of Arabidopsis and rice. Prefixes:At, Arabidopsis thaliana; Gh, Gossypium herbaceum; Lj, Lotus japonicus;Mt, Medicago truncatula; Na, Nicotiana alata; Os, Oriza sativa; Pc,Populus canescens; Ptr, Populus tremula ×tremuloides; Ze, Zinniaelegans; Zm, Zea mays.

FIG. 5: Expression pattern of CesA10, 11 and 12 in different maizetissues. All three genes are nearly synchronously expressed in tissuesrich in secondary wall.

FIG. 6: Effect of overexpression of different CesA genes on plant heightin corn. Whereas the overexpression of CesA8 led to an increase inheight, CesA4 and CesA5 had not effect. Overexpression of CesA1 resultedin stunted plants.

FIG. 7: Effect of the overexpression of different CesA genes on theamount of cellulose in a unit length of the stalk tissue below ear incorn. CesA4 and CesA8, when overexpressed, resulted in an increasedcellulose/length, CesA5 had no effect and CesA1 resulted in reducedcellulose/length.

FIG. 8: Contribution of different stalk components to dry matter,diameter, volume, and stalk strength in maize hybrids. The data arederived from seven hybrids grown at three densities (27, 43 and 59 K peracre) in three replications each in 2001. Two stalks were sampled fromeach replication. Internodes 3 and 4 below the ear were broken withInstron model 4411 (Instron Corporation, 100 Royall Street, Canton,Mass. 02021). After breaking, the 3rd internode was separated into rindand inner tissue. Path coefficient analyses were performed using rindand inner tissue as independent variables (X₁ and X₂, respectively) andthe whole stalk as the dependent variable (Y). The multiple regressionequation: Y=a+b₁X₁+b₂X₂+e where a is the intercept and e error. Pathcoefficients were calculated as follows: ρYXn=b_(n)*δ_(n)/δ_(Y) where nis 1 or 2. The contribution of each independent variable to whole stalk(Y) was calculated as follows: ρYxn*rYxn where r is the correlationcoefficient.

FIG. 9: Expression of the maize CesA genes in the pulvinal tissue ofleaf derived from an elongating internode. The expression was studied bythe Lynx MPSS technology.

DETAILED DESCRIPTION OF THE INVENTION Overview

A. Nucleic Acids and Protein of the Present Invention

Unless otherwise stated, the polynucleotide and polypeptide sequencesidentified in Table 1 represent polynucleotides and polypeptides of thepresent invention. Table 1 cross-references these polynucleotide andpolypeptides to their gene name and internal database identificationnumber (SEQ ID NO.). A nucleic acid of the present invention comprises apolynucleotide of the present invention. A protein of the presentinvention comprises a polypeptide of the present invention.

TABLE 1 Database ID Polynucleotide Polypeptide SEQ Gene Name NO: SEQ IDNO: ID NO: Cellulose synthase CesA-1 1 2 Cellulose synthase CesA-2 45 46Cellulose synthase CesA-3 5 6 Cellulose synthase CesA-4 9 10 Cellulosesynthase CesA-5 13 14 Cellulose synthase CesA-6 41 42 Cellulose synthaseCesA-7 49 50 Cellulose synthase CesA-8 17 18 Cellulose synthase CesA-921 22 Cellulose synthase CesA-10 25 26 Cellulose synthase CesA-11 27 28Cellulose synthase CesA-12 29 30

Table 2 further provides a comparison detailing the homology as apercentage of the 12 CesA genes from maize that have been describedherein (see, also, “Related Applications” above).

TABLE 2 CesA1 CesA2 CesA3 CesA4 CesA5 CesA6 CesA7 CesA8 CesA9 CesA10CesA11 CesA12 CesA1 93 60 59 60 55 55 57 61 51 51 46 CesA2 60 59 61 5555 57 61 51 51 47 CesA3 47 48 49 45 46 49 46 52 50 CesA4 77 54 52 58 8654 53 52 CesA5 55 53 57 75 52 52 51 CesA6 74 73 56 56 55 53 CesA7 70 5450 48 46 CesA8 59 55 52 51 CesA9 52 52 50 CesA10 53 64 CesA11 56 CesA12

Further characterization of the CesA group is provided in FIG. 4, as aconsensus tree for plant Ces A proteins. It describes the relationshipbetween Ces A from maize, rice and Arabidopsis sources.

B. Exemplary Utility of the Present Invention

The present invention provides utility in such exemplary applications asimprovement of stalk quality for improved stand lodging or standabilityor silage digestibility. Further, the present invention provides for anincreased concentration of cellulose in the pericarp, hardening thekernel and thus improving its handling ability. Stalk lodging atmaturity can cause significant yield losses in corn. Environmentalstresses from flowering to harvest, such as drought and nutrientdeficiency, further worsen this problem. The effect of abiotic stressesis exacerbated by biotic factors, such as stalk rot resulting from thesoil-living pathogens growing through the ground tissue.

Maize hybrids known to be resistant to stalk lodging have mechanicallystronger stalks. At the compositional level, cellulose in a unit stalklength is highly correlated with breaking strength. The presentinvention provides for modulation of cellulose synthase compositionleading to increased stalk strength.

Definitions

Units, prefixes and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges recitedwithin the specification are inclusive of the numbers defining the rangeand include each integer within the defined range. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by the one-letter symbols recommended by the IUPAC-IUBMB NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. Unless otherwise provided for, software,electrical, and electronics terms as used herein are as defined in TheNew IEEE Standard Dictionary of Electrical and Electronics Terms (5^(th)edition, 1993). The terms defined below are more fully defined byreference to the specification as a whole. Section headings providedthroughout the specification are not limitations to the various objectsand embodiments of the present invention.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), O-Beta Replicasesystems, transcription-based amplification system (TAS) and stranddisplacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, Persing, et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

As used herein, “antisense orientation” includes reference to a duplexpolynucleotide sequence that is operably linked to a promoter in anorientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as are present in some plant, animal and fungalmitochondria, the bacterium Mycoplasma capricolumn or the ciliateMacronucleus, may be used when the nucleic acid is expressed therein.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray, et al., (1989) Nucl. Acids Res.17:477-498). Thus, the maize preferred codon for a particular amino acidmay be derived from known gene sequences from maize. Maize codon usagefor 28 genes from maize plants is listed in Table 4 of Murray, et al.,supra.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of a native (non-synthetic), endogenous, biologically (e.g.,structurally or catalytically) active form of the specified protein.Methods to determine whether a sequence is full-length are well known inthe art, including such exemplary techniques as northern or westernblots, primer extension, S1 protection and ribonuclease protection. See,e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Comparison to known full-lengthhomologous (orthologous and/or paralogous) sequences can also be used toidentify full-length sequences of the present invention. Additionally,consensus sequences typically present at the 5′ and 3′ untranslatedregions of mRNA aid in the identification of a polynucleotide asfull-length. For example, the consensus sequence ANNNNAUGG, where theunderlined codon represents the N-terminal methionine, aids indetermining whether the polynucleotide has a complete 5′ end. Consensussequences at the 3′ end, such as polyadenylation sequences, aid indetermining whether the polynucleotide has a complete 3′ end.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by human intervention. For example, apromoter operably linked to a heterologous structural gene is from aspecies different from that from which the structural gene was derived,or, if from the same species, one or both are substantially modifiedfrom their original form. A heterologous protein may originate from aforeign species or, if from the same species, is substantially modifiedfrom its original form by human intervention.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “introduced” includes reference to the incorporation of anucleic acid into a eukaryotic or prokaryotic cell where the nucleicacid may be incorporated into the genome of the cell (e.g., chromosome,plasmid, plastid or mitochondrial DNA), converted into an autonomousreplicon, or transiently expressed (e.g., transfected mRNA). The termincludes such nucleic acid introduction means as “transfection”,“transformation” and “transduction”.

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with it as found in its naturalenvironment. The isolated material optionally comprises material notfound with the material in its natural environment or (2) if thematerial is in its natural environment, the material has beensynthetically altered or synthetically produced by deliberate humanintervention and/or placed at a different location within the cell. Thesynthetic alteration or creation of the material can be performed on thematerial within or apart from its natural state. For example, anaturally-occurring nucleic acid becomes an isolated nucleic acid if itis altered or produced by non-natural, synthetic methods or if it istranscribed from DNA which has been altered or produced by non-natural,synthetic methods. The isolated nucleic acid may also be produced by thesynthetic re-arrangement (“shuffling”) of a part or parts of one or moreallelic forms of the gene of interest. Likewise, a naturally-occurringnucleic acid (e.g., a promoter) becomes isolated if it is introduced toa different locus of the genome. Nucleic acids which are “isolated,” asdefined herein, are also referred to as “heterologous” nucleic acids.See, e.g., Compounds and Methods for Site Directed Mutagenesis inEukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo HomologousSequence Targeting in Eukaryotic Cells, Zarling, et al., WO 93/22443(PCT/US93/03868).

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer, or chimeras thereof, ineither single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism, tissue or of acell type from that organism. Construction of exemplary nucleic acidlibraries, such as genomic and cDNA libraries, is taught in standardmolecular biology references such as Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol. 152, AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook, et al., MolecularCloning—A Laboratory Manual, 2^(nd) ed., Vol. 1-3 (1989) and CurrentProtocols in Molecular Biology, Ausubel, et al., Eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (1994).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

As used herein, the term “plant” includes reference to whole plants,plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells,seeds and progeny of same. Plant cell, as used herein, further includes,without limitation, cells obtained from or found in: seeds, suspensioncultures, embryos, meristematic regions, callus tissue, leaves, roots,shoots, gametophytes, sporophytes, pollen and microspores. Plant cellscan also be understood to include modified cells, such as protoplasts,obtained from the aforementioned tissues. The class of plants which canbe used in the methods of the invention is generally as broad as theclass of higher plants amenable to transformation techniques, includingboth monocotyledonous and dicotyledonous plants. A particularlypreferred plant is Zea mays.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide or chimeras or analogsthereof that have the essential nature of a natural deoxy- orribo-nucleotide in that they hybridize, under stringent hybridizationconditions, to substantially the same nucleotide sequence as naturallyoccurring nucleotides and/or allow translation into the same aminoacid(s) as the naturally occurring nucleotide(s). A polynucleotide canbe full-length or a subsequence of a native or heterologous structuralor regulatory gene. Unless otherwise indicated, the term includesreference to the specified sequence as well as the complementarysequence thereof. Thus, DNAs or RNAs with backbones modified forstability or for other reasons are “polynucleotides” as that term isintended herein. Moreover, DNAs or RNAs comprising unusual bases, suchas inosine, or modified bases, such as tritylated bases, to name justtwo examples, are polynucleotides as the term is used herein. It will beappreciated that a great variety of modifications have been made to DNAand RNA that serve many useful purposes known to those of skill in theart. The term polynucleotide as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. Further, this invention contemplatesthe use of both the methionine-containing and the methionine-less aminoterminal variants of the protein of the invention.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses and bacteria which comprise genes expressed inplant cells such Agrobacterium or Rhizobium. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots or seeds. Suchpromoters are referred to as “tissue preferred”. Promoters whichinitiate transcription only in certain tissue are referred to as “tissuespecific”. A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “repressible” promoter is apromoter which is under environmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue specific, tissuepreferred, cell type specific and inducible promoters constitute theclass of “non-constitutive” promoters. A “constitutive” promoter is apromoter which is active under most environmental conditions.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all as a result of human intervention. The term“recombinant” as used herein does not encompass the alteration of thecell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed and apromoter.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass non-natural analogs of natural amino acids thatcan function in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, preferably 90% sequenceidentity and most preferably 100% sequence identity (i.e.,complementary) with each other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence, to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C. and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, (1984) Anal. Biochem., 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point(“T_(m)”) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than theT_(m); moderately stringent conditions can utilize a hybridizationand/or wash at 6, 7, 8, 9 or 10° C. lower than the T_(m); low stringencyconditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15or 20° C. lower than the T_(m). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. Hybridizationand/or wash conditions can be applied for at least 10, 30, 60, 90, 120or 240 minutes. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

As used herein, “transgenic plant” includes reference to a plant whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used inintroduction of a polynucleotide of the present invention into a hostcell. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween a polynucleotide/polypeptide of the present invention with areference polynucleotide/polypeptide: (a) “reference sequence”, (b)“comparison window”, (c) “sequence identity” and (d) “percentage ofsequence identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison with a polynucleotide/polypeptide of thepresent invention. A reference sequence may be a subset or the entiretyof a specified sequence; for example, as a segment of a full-length cDNAor gene sequence or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” includes reference to acontiguous and specified segment of a polynucleotide/polypeptidesequence, wherein the polynucleotide/polypeptide sequence may becompared to a reference sequence and wherein the portion of thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acids residues in length, andoptionally can be 30, 40, 50, 100 or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide/polypeptide sequence, a gappenalty is typically introduced and is subtracted from the number ofmatches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, (1981) Adv. Appl.Math. 2:482; by the homology alignment algorithm of Needleman andWunsch, (1970) J. Mol. Biol. 48:443; by the search for similarity methodof Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA and TFASTA in the WisconsinGenetics Software Package®, Genetics Computer Group (GCG®), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, (1988) Gene 73:237-244; Higgins and Sharp, (1989)CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Research16:10881-90; Huang, et al., (1992) Computer Applications in theBiosciences 8:155-65 and Pearson, et al., (1994) Methods in MolecularBiology 24:307-331.

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, Current Protocolsin Molecular Biology, Chapter 19, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995); Altschul, et al.,(1990) J. Mol. Biol., 215:403-410 and Altschul, et al., (1997) NucleicAcids Res. 25:3389-3402.

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold. These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see, Henikoff and Henikoff, (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul, (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5877). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequenceswhich may be homopolymeric tracts, short-period repeats or regionsenriched in one or more amino acids. Such low-complexity regions may bealigned between unrelated proteins even though other regions of theprotein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-163)and XNU (Claverie and States, (1993) Comput. Chem. 17:191-201)low-complexity filters can be employed alone or in combination.

Unless otherwise stated, nucleotide and protein identity/similarityvalues provided herein are calculated using GAP (GCG® Version 10) underdefault values.

GAP (Global Alignment Program) can also be used to compare apolynucleotide or polypeptide of the present invention with a referencesequence. GAP uses the algorithm of Needleman and Wunsch, (J. Mol. Biol.48: 443-453 (1970)) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package® for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 100. Thus, for example, the gapcreation and gap extension penalties can each independently be: 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the Wisconsin Genetics Software Package® isBLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA89:10915).

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp, (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci.4:11-17 e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

Utilities

The present invention provides, among other things, compositions andmethods for modulating (i.e., increasing or decreasing) the level ofpolynucleotides and polypeptides of the present invention in plants. Inparticular, the polynucleotides and polypeptides of the presentinvention can be expressed temporally or spatially, e.g., atdevelopmental stages, in tissues and/or in quantities, which areuncharacteristic of non-recombinantly engineered plants.

The present invention also provides isolated nucleic acids comprisingpolynucleotides of sufficient length and complementarity to apolynucleotide of the present invention to use as probes oramplification primers in the detection, quantitation or isolation ofgene transcripts. For example, isolated nucleic acids of the presentinvention can be used as probes in detecting deficiencies in the levelof mRNA in screenings for desired transgenic plants, for detectingmutations in the gene (e.g., substitutions, deletions or additions), formonitoring upregulation of expression or changes in enzyme activity inscreening assays of compounds, for detection of any number of allelicvariants (polymorphisms), orthologs or paralogs of the gene or for sitedirected mutagenesis in eukaryotic cells (see, e.g., U.S. Pat. No.5,565,350). The isolated nucleic acids of the present invention can alsobe used for recombinant expression of their encoded polypeptides or foruse as immunogens in the preparation and/or screening of antibodies. Theisolated nucleic acids of the present invention can also be employed foruse in sense or antisense suppression of one or more genes of thepresent invention in a host cell, tissue or plant. Attachment ofchemical agents which bind, intercalate, cleave and/or crosslink to theisolated nucleic acids of the present invention can also be used tomodulate transcription or translation.

The present invention also provides isolated proteins comprising apolypeptide of the present invention (e.g., preproenzyme, proenzyme orenzymes). The present invention also provides proteins comprising atleast one epitope from a polypeptide of the present invention. Theproteins of the present invention can be employed in assays for enzymeagonists or antagonists of enzyme function or for use as immunogens orantigens to obtain antibodies specifically immunoreactive with a proteinof the present invention. Such antibodies can be used in assays forexpression levels, for identifying and/or isolating nucleic acids of thepresent invention from expression libraries, for identification ofhomologous polypeptides from other species or for purification ofpolypeptides of the present invention.

The isolated nucleic acids and polypeptides of the present invention canbe used over a broad range of plant types, particularly monocots such asthe species of the family Gramineae including Hordeum, Secale, Oryza,Triticum, Sorghum (e.g., S. bicolor) and Zea (e.g., Z. mays) and dicotssuch as Glycine.

The isolated nucleic acid and proteins of the present invention can alsobe used in species from the genera: Cucurbita, Rosa, Vitis, Juglans,Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna,Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis,Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis, Browallia, Pisum, Phaseolus, Lolium and Avena.

Nucleic Acids

The present invention provides, among other things, isolated nucleicacids of RNA, DNA and analogs and/or chimeras thereof, comprising apolynucleotide of the present invention.

A polynucleotide of the present invention is inclusive of those in Table1 and:

(a) an isolated polynucleotide encoding a polypeptide of the presentinvention such as those referenced in Table 1, including exemplarypolynucleotides of the present invention;

(b) an isolated polynucleotide which is the product of amplificationfrom a plant nucleic acid library using primer pairs which selectivelyhybridize under stringent conditions to loci within a polynucleotide ofthe present invention;

(c) an isolated polynucleotide which selectively hybridizes to apolynucleotide of (a) or (b);

(d) an isolated polynucleotide having a specified sequence identity withpolynucleotides of (a), (b) or (c);

(e) an isolated polynucleotide encoding a protein having a specifiednumber of contiguous amino acids from a prototype polypeptide, whereinthe protein is specifically recognized by antisera elicited bypresentation of the protein and wherein the protein does not detectablyimmunoreact to antisera which has been fully immunosorbed with theprotein;

(f) complementary sequences of polynucleotides of (a), (b), (c), (d) or(e);

(g) an isolated polynucleotide comprising at least a specific number ofcontiguous nucleotides from a polynucleotide of (a), (b), (c), (d), (e)or (f);

(h) an isolated polynucleotide from a full-length enriched cDNA libraryhaving the physico-chemical property of selectively hybridizing to apolynucleotide of (a), (b), (c), (d), (e), (f) or (g);

(i) an isolated polynucleotide made by the process of: 1) providing afull-length enriched nucleic acid library, 2) selectively hybridizingthe polynucleotide to a polynucleotide of (a), (b), (c), (d), (e), (f),(g) or (h), thereby isolating the polynucleotide from the nucleic acidlibrary.

A. Polynucleotides Encoding A Polypeptide of the Present Invention

As indicated in (a), above, the present invention provides isolatednucleic acids comprising a polynucleotide of the present invention,wherein the polynucleotide encodes a polypeptide of the presentinvention. Every nucleic acid sequence herein that encodes a polypeptidealso, by reference to the genetic code, describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine and UGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Thus, each silent variation of a nucleic acid which encodes apolypeptide of the present invention is implicit in each describedpolypeptide sequence and is within the scope of the present invention.Accordingly, the present invention includes polynucleotides of thepresent invention and polynucleotides encoding a polypeptide of thepresent invention.

B. Polynucleotides Amplified from a Plant Nucleic Acid Library

As indicated in (b), above, the present invention provides an isolatednucleic acid comprising a polynucleotide of the present invention,wherein the polynucleotides are amplified, under nucleic acidamplification conditions, from a plant nucleic acid library. Nucleicacid amplification conditions for each of the variety of amplificationmethods are well known to those of ordinary skill in the art. The plantnucleic acid library can be constructed from a monocot such as a cerealcrop. Exemplary cereals include maize, sorghum, alfalfa, canola, wheator rice. The plant nucleic acid library can also be constructed from adicot such as soybean. Zea mays lines B73, PHRE1, A632, BMS-P2#10, W23and Mo17 are known and publicly available. Other publicly known andavailable maize lines can be obtained from the Maize GeneticsCooperation (Urbana, Ill.). Wheat lines are available from the WheatGenetics Resource Center (Manhattan, Kans.).

The nucleic acid library may be a cDNA library, a genomic library or alibrary generally constructed from nuclear transcripts at any stage ofintron processing. cDNA libraries can be normalized to increase therepresentation of relatively rare cDNAs. In optional embodiments, thecDNA library is constructed using an enriched full-length cDNA synthesismethod. Examples of such methods include Oligo-Capping (Maruyama andSugano, (1994) Gene 138:171-174), Biotinylated CAP Trapper (Carninci, etal., (1996) Genomics 37:327-336) and CAP Retention Procedure (Edery, etal., (1995) Molecular and Cellular Biology 15:3363-3371). Rapidlygrowing tissues or rapidly dividing cells are preferred for use as anmRNA source for construction of a cDNA library. Growth stages of maizeare described in “How a Corn Plant Develops,” Special Report Number 48,Iowa State University of Science and Technology Cooperative ExtensionService, Ames, Iowa, Reprinted February 1993.

A polynucleotide of this embodiment (or subsequences thereof) can beobtained, for example, by using amplification primers which areselectively hybridized and primer extended, under nucleic acidamplification conditions, to at least two sites within a polynucleotideof the present invention, or to two sites within the nucleic acid whichflank and comprise a polynucleotide of the present invention, or to asite within a polynucleotide of the present invention and a site withinthe nucleic acid which comprises it. Methods for obtaining 5′ and/or 3′ends of a vector insert are well known in the art. See, e.g., RACE(Rapid Amplification of Complementary Ends) as described in Frohman, inPCR Protocols: A Guide to Methods and Applications, Innis, et al., Eds.(Academic Press, Inc., San Diego), pp. 28-38 (1990)); see, also, U.S.Pat. No. 5,470,722 and Current Protocols in Molecular Biology, Unit15.6, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience,New York (1995); Frohman and Martin, Techniques 1:165 (1989).

Optionally, the primers are complementary to a subsequence of the targetnucleic acid which they amplify but may have a sequence identity rangingfrom about 85% to 99% relative to the polynucleotide sequence which theyare designed to anneal to. As those skilled in the art will appreciate,the sites to which the primer pairs will selectively hybridize arechosen such that a single contiguous nucleic acid can be formed underthe desired nucleic acid amplification conditions. The primer length innucleotides is selected from the group of integers consisting of from atleast 15 to 50. Thus, the primers can be at least 15, 18, 20, 25, 30, 40or 50 nucleotides in length. Those of skill will recognize that alengthened primer sequence can be employed to increase specificity ofbinding (i.e., annealing) to a target sequence. A non-annealing sequenceat the 5′ end of a primer (a “tail”) can be added, for example, tointroduce a cloning site at the terminal ends of the amplicon.

The amplification products can be translated using expression systemswell known to those of skill in the art. The resulting translationproducts can be confirmed as polypeptides of the present invention by,for example, assaying for the appropriate catalytic activity (e.g.,specific activity and/or substrate specificity) or verifying thepresence of one or more epitopes which are specific to a polypeptide ofthe present invention. Methods for protein synthesis from PCR derivedtemplates are known in the art and available commercially. See, e.g.,Amersham Life Sciences, Inc, Catalog '97, p. 354.

C. Polynucleotides Which Selectively Hybridize to a Polynucleotide of(A) or (B)

As indicated in (c), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides selectively hybridize, under selectivehybridization conditions, to a polynucleotide of sections (A) or (B) asdiscussed above. Thus, the polynucleotides of this embodiment can beused for isolating, detecting, and/or quantifying nucleic acidscomprising the polynucleotides of (A) or (B). For example,polynucleotides of the present invention can be used to identify,isolate, or amplify partial or full-length clones in a depositedlibrary. In some embodiments, the polynucleotides are genomic or cDNAsequences isolated or otherwise complementary to a cDNA from a dicot ormonocot nucleic acid library. Exemplary species of monocots and dicotsinclude, but are not limited to: maize, canola, soybean, cotton, wheat,sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley and rice.The cDNA library comprises at least 50% to 95% full-length sequences(for example, at least 50%, 60%, 70%, 80%, 90% or 95% full-lengthsequences). The cDNA libraries can be normalized to increase therepresentation of rare sequences. See, e.g., U.S. Pat. No. 5,482,845.Low stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% to 80% sequence identity and can be employed toidentify orthologous or paralogous sequences.

D. Polynucleotides Having a Specific Sequence Identity with thePolynucleotides of (A), (B) or (C)

As indicated in (d), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides have a specified identity at the nucleotidelevel to a polynucleotide as disclosed above in sections (A), (B) or(C), above. Identity can be calculated using, for example, the BLAST,CLUSTALW or GAP algorithms under default conditions. The percentage ofidentity to a reference sequence is at least 50% and, rounded upwards tothe nearest integer, can be expressed as an integer selected from thegroup of integers consisting of from 50 to 99. Thus, for example, thepercentage of identity to a reference sequence can be at least 60%, 70%,75%, 80%, 85%, 90% or 95%.

Optionally, the polynucleotides of this embodiment will encode apolypeptide that will share an epitope with a polypeptide encoded by thepolynucleotides of sections (A), (B) or (C). Thus, these polynucleotidesencode a first polypeptide which elicits production of antiseracomprising antibodies which are specifically reactive to a secondpolypeptide encoded by a polynucleotide of (A), (B) or (C). However, thefirst polypeptide does not bind to antisera raised against itself whenthe antisera has been fully immunosorbed with the first polypeptide.Hence, the polynucleotides of this embodiment can be used to generateantibodies for use in, for example, the screening of expressionlibraries for nucleic acids comprising polynucleotides of (A), (B) or(C), or for purification of, or in immunoassays for, polypeptidesencoded by the polynucleotides of (A), (B) or (C). The polynucleotidesof this embodiment comprise nucleic acid sequences which can be employedfor selective hybridization to a polynucleotide encoding a polypeptideof the present invention.

Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure. Antibody screening ofpeptide display libraries is well known in the art. The displayedpeptide sequences can be from 3 to 5000 or more amino acids in length,frequently from 5-100 amino acids long, and often from about 8 to 15amino acids long. In addition to direct chemical synthetic methods forgenerating peptide libraries, several recombinant DNA methods have beendescribed. One type involves the display of a peptide sequence on thesurface of a bacteriophage or cell. Each bacteriophage or cell containsthe nucleotide sequence encoding the particular displayed peptidesequence. Such methods are described in PCT Patent Publication Numbers91/17271, 91/18980, 91/19818 and 93/08278. Other systems for generatinglibraries of peptides have aspects of both in vitro chemical synthesisand recombinant methods. See, PCT Patent Publication Numbers 92/05258,92/14843 and 97/20078. See also, U.S. Pat. Nos. 5,658,754 and 5,643,768.Peptide display libraries, vectors, and screening kits are commerciallyavailable from such suppliers as Invitrogen (Carlsbad, Calif.).

E. Polynucleotides Encoding a Protein Having a Subsequence from aPrototype Polypeptide and Cross-Reactive to the Prototype Polypeptide

As indicated in (e), above, the present invention provides isolatednucleic acids comprising polynucleotides of the present invention,wherein the polynucleotides encode a protein having a subsequence ofcontiguous amino acids from a prototype polypeptide of the presentinvention such as are provided in (a), above. The length of contiguousamino acids from the prototype polypeptide is selected from the group ofintegers consisting of from at least 10 to the number of amino acidswithin the prototype sequence. Thus, for example, the polynucleotide canencode a polypeptide having a subsequence having at least 10, 15, 20,25, 30, 35, 40, 45 or 50, contiguous amino acids from the prototypepolypeptide. Further, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4 or 5. Thesubsequences can be separated by any integer of nucleotides from 1 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100 or 200 nucleotides.

The proteins encoded by polynucleotides of this embodiment, whenpresented as an immunogen, elicit the production of polyclonalantibodies which specifically bind to a prototype polypeptide such asbut not limited to, a polypeptide encoded by the polynucleotide of (a)or (b), above. Generally, however, a protein encoded by a polynucleotideof this embodiment does not bind to antisera raised against theprototype polypeptide when the antisera has been fully immunosorbed withthe prototype polypeptide. Methods of making and assaying for antibodybinding specificity/affinity are well known in the art. Exemplaryimmunoassay formats include ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

In a preferred assay method, fully immunosorbed and pooled antiserawhich is elicited to the prototype polypeptide can be used in acompetitive binding assay to test the protein. The concentration of theprototype polypeptide required to inhibit 50% of the binding of theantisera to the prototype polypeptide is determined. If the amount ofthe protein required to inhibit binding is less than twice the amount ofthe prototype protein, then the protein is said to specifically bind tothe antisera elicited to the immunogen. Accordingly, the proteins of thepresent invention embrace allelic variants, conservatively modifiedvariants and minor recombinant modifications to a prototype polypeptide.

A polynucleotide of the present invention optionally encodes a proteinhaving a molecular weight as the non-glycosylated protein within 20% ofthe molecular weight of the full-length non-glycosylated polypeptides ofthe present invention. Molecular weight can be readily determined bySDS-PAGE under reducing conditions. Optionally, the molecular weight iswithin 15% of a full length polypeptide of the present invention, morepreferably within 10% or 5%, and most preferably within 3%, 2% or 1% ofa full length polypeptide of the present invention.

Optionally, the polynucleotides of this embodiment will encode a proteinhaving a specific enzymatic activity at least 50%, 60%, 80% or 90% of acellular extract comprising the native, endogenous full-lengthpolypeptide of the present invention. Further, the proteins encoded bypolynucleotides of this embodiment will optionally have a substantiallysimilar affinity constant (K_(m)) and/or catalytic activity (i.e., themicroscopic rate constant, k_(cat)) as the native endogenous,full-length protein. Those of skill in the art will recognize thatk_(cat)/K_(m) value determines the specificity for competing substratesand is often referred to as the specificity constant. Proteins of thisembodiment can have a k_(cat)/K_(m) value at least 10% of a full-lengthpolypeptide of the present invention as determined using the endogenoussubstrate of that polypeptide. Optionally, the k_(cat)/K_(m) value willbe at least 20%, 30%, 40%, 50% and most preferably at least 60%, 70%,80%, 90% or 95% the k_(cat)/K_(m) value of the full-length polypeptideof the present invention. Determination of k_(cat), K_(m), andk_(cat)/K_(m) can be determined by any number of means well known tothose of skill in the art. For example, the initial rates (i.e., thefirst 5% or less of the reaction) can be determined using rapid mixingand sampling techniques (e.g., continuous-flow, stopped-flow or rapidquenching techniques), flash photolysis or relaxation methods (e.g.,temperature jumps) in conjunction with such exemplary methods ofmeasuring as spectrophotometry, spectrofluorimetry, nuclear magneticresonance or radioactive procedures. Kinetic values are convenientlyobtained using a Lineweaver-Burk or Eadie-Hofstee plot.

F. Polynucleotides Complementary to the Polynucleotides of (A)-(E)

As indicated in (f), above, the present invention provides isolatednucleic acids comprising polynucleotides complementary to thepolynucleotides of paragraphs A-E, above. As those of skill in the artwill recognize, complementary sequences base-pair throughout theentirety of their length with the polynucleotides of sections (A)-(E)(i.e., have 100% sequence identity over their entire length).Complementary bases associate through hydrogen bonding in doublestranded nucleic acids. For example, the following base pairs arecomplementary: guanine and cytosine; adenine and thymine and adenine anduracil.

G. Polynucleotides Which are Subsequences of the Polynucleotides of(A)-(F)

As indicated in (g), above, the present invention provides isolatednucleic acids comprising polynucleotides which comprise at least 15contiguous bases from the polynucleotides of sections (A) through (F) asdiscussed above. The length of the polynucleotide is given as an integerselected from the group consisting of from at least 15 to the length ofthe nucleic acid sequence from which the polynucleotide is a subsequenceof. Thus, for example, polynucleotides of the present invention areinclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50,60, 75 or 100 contiguous nucleotides in length from the polynucleotidesof (A)-(F). Optionally, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4 or 5. Thesubsequences can be separated by any integer of nucleotides from 1 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100 or 200 nucleotides.

Subsequences can be made by in vitro synthetic, in vitro biosynthetic orin vivo recombinant methods. In optional embodiments, subsequences canbe made by nucleic acid amplification. For example, nucleic acid primerswill be constructed to selectively hybridize to a sequence (or itscomplement) within, or co-extensive with, the coding region.

The subsequences of the present invention can comprise structuralcharacteristics of the sequence from which it is derived. Alternatively,the subsequences can lack certain structural characteristics of thelarger sequence from which it is derived such as a poly(A) tail.Optionally, a subsequence from a polynucleotide encoding a polypeptidehaving at least one epitope in common with a prototype polypeptidesequence as provided in (a), above, may encode an epitope in common withthe prototype sequence. Alternatively, the subsequence may not encode anepitope in common with the prototype sequence but can be used to isolatethe larger sequence by, for example, nucleic acid hybridization with thesequence from which it's derived. Subsequences can be used to modulateor detect gene expression by introducing into the subsequences compoundswhich bind, intercalate, cleave and/or crosslink to nucleic acids.Exemplary compounds include acridine, psoralen, phenanthroline,naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.

H. Polynucleotides From a Full-Length Enriched cDNA Library Having thePhysico-Chemical Property of Selectively Hybridizing to a Polynucleotideof (A)-(G)

As indicated in (h), above, the present invention provides an isolatedpolynucleotide from a full-length enriched cDNA library having thephysico-chemical property of selectively hybridizing to a polynucleotideof paragraphs (A), (B), (C), (D), (E), (F) or (G) as discussed above.Methods of constructing full-length enriched cDNA libraries are known inthe art and discussed briefly below. The cDNA library comprises at least50% to 95% full-length sequences (for example, at least 50%, 60%, 70%,80%, 90% or 95% full-length sequences). The cDNA library can beconstructed from a variety of tissues from a monocot or dicot at avariety of developmental stages. Exemplary species include maize, wheat,rice, canola, soybean, cotton, sorghum, sunflower, alfalfa, oats, sugarcane, millet, barley and rice. Methods of selectively hybridizing, underselective hybridization conditions, a polynucleotide from a full-lengthenriched library to a polynucleotide of the present invention are knownto those of ordinary skill in the art. Any number of stringencyconditions can be employed to allow for selective hybridization. Inoptional embodiments, the stringency allows for selective hybridizationof sequences having at least 70%, 75%, 80%, 85%, 90%, 95% or 98%sequence identity over the length of the hybridized region. Full-lengthenriched cDNA libraries can be normalized to increase the representationof rare sequences.

I Polynucleotide Products Made by a cDNA Isolation Process

As indicated in (I), above, the present invention provides an isolatedpolynucleotide made by the process of: 1) providing a full-lengthenriched nucleic acid library, 2) selectively hybridizing thepolynucleotide to a polynucleotide of paragraphs (A), (B), (C), (D),(E), (F), (G) or (H) as discussed above, and thereby isolating thepolynucleotide from the nucleic acid library. Full-length enrichednucleic acid libraries are constructed as discussed in paragraph (G) andbelow. Selective hybridization conditions are as discussed in paragraph(G). Nucleic acid purification procedures are well known in the art.Purification can be conveniently accomplished using solid-phase methods;such methods are well known to those of skill in the art and kits areavailable from commercial suppliers such as Advanced Biotechnologies(Surrey, UK). For example, a polynucleotide of paragraphs (A)-(H) can beimmobilized to a solid support such as a membrane, bead, or particle.See, e.g., U.S. Pat. No. 5,667,976. The polynucleotide product of thepresent process is selectively hybridized to an immobilizedpolynucleotide and the solid support is subsequently isolated fromnon-hybridized polynucleotides by methods including, but not limited to,centrifugation, magnetic separation, filtration, electrophoresis and thelike.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified or otherwise constructedfrom a monocot such as maize, rice or wheat or a dicot such as soybean.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. A polynucleotideof the present invention can be attached to a vector, adapter or linkerfor cloning and/or expression of a polynucleotide of the presentinvention. Additional sequences may be added to such cloning and/orexpression sequences to optimize their function in cloning and/orexpression, to aid in isolation of the polynucleotide, or to improve theintroduction of the polynucleotide into a cell. Typically, the length ofa nucleic acid of the present invention less the length of itspolynucleotide of the present invention is less than 20 kilobase pairs,often less than 15 kb and frequently less than 10 kb. Use of cloningvectors, expression vectors, adapters, and linkers is well known andextensively described in the art. For a description of various nucleicacids see, for example, Stratagene Cloning Systems, Catalogs 1999 (LaJolla, Calif.) and Amersham Life Sciences, Inc, Catalog '99 (ArlingtonHeights, Ill.).

A. Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. Isolation of RNA, andconstruction of cDNA and genomic libraries is well known to those ofordinary skill in the art. See, e.g., Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and,Current Protocols in Molecular Biology, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

A1. Full-Length Enriched cDNA Libraries

A number of cDNA synthesis protocols have been described which provideenriched full-length cDNA libraries. Enriched full-length cDNA librariesare constructed to comprise at least 600%, and more preferably at least70%, 80%, 90% or 95% full-length inserts amongst clones containinginserts. The length of insert in such libraries can be at least 2, 3, 4,5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors to accommodate insertsof these sizes are known in the art and available commercially. See,e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12kb cloning capacity). An exemplary method of constructing a greater than95% pure full-length cDNA library is described by Carninci, et al.,(1996) Genomics, 37:327-336. Other methods for producing full-lengthlibraries are known in the art. See, e.g., Edery, et al., (1995) Mol.Cell. Biol. 15(6):3363-3371 and PCT Application Number WO 96/34981.

A2 Normalized or Subtracted cDNA Libraries

A non-normalized cDNA library represents the mRNA population of thetissue it was made from. Since unique clones are out-numbered by clonesderived from highly expressed genes their isolation can be laborious.Normalization of a cDNA library is the process of creating a library inwhich each clone is more equally represented. Construction of normalizedlibraries is described in Ko, (1990) Nucl. Acids. Res. 18(19):5705-5711;Patanjali, et al., (1991) Proc. Natl. Acad. U.S.A. 88:1943-1947; U.S.Pat. Nos. 5,482,685, 5,482,845 and 5,637,685. In an exemplary methoddescribed by Soares, et al., normalization resulted in reduction of theabundance of clones from a range of four orders of magnitude to a narrowrange of only 1 order of magnitude. Proc. Natl. Acad. Sci. USA,91:9228-9232 (1994).

Subtracted cDNA libraries are another means to increase the proportionof less abundant cDNA species. In this procedure, cDNA prepared from onepool of mRNA is depleted of sequences present in a second pool of mRNAby hybridization. The cDNA:mRNA hybrids are removed and the remainingun-hybridized cDNA pool is enriched for sequences unique to that pool.See, Foote, et al., in, Plant Molecular Biology: A Laboratory Manual,Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, (1991)Technique 3(2):58-63; Sive and St. John, (1988) Nucl. Acids Res.,16(22):10937; Current Protocols in Molecular Biology, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995) andSwaroop, et al., (1991) Nucl. Acids Res., 19(8):1954. cDNA subtractionkits are commercially available. See, e.g., PCR-Select (Clontech, PaloAlto, Calif.).

To construct genomic libraries, large segments of genomic DNA aregenerated by fragmentation, e.g., using restriction endonucleases, andare ligated with vector DNA to form concatemers that can be packagedinto the appropriate vector. Methodologies to accomplish these ends andsequencing methods to verify the sequence of nucleic acids are wellknown in the art. Examples of appropriate molecular biologicaltechniques and instructions sufficient to direct persons of skillthrough many construction, cloning and screening methodologies are foundin Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods in Enzymology,Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel,Eds., San Diego: Academic Press, Inc. (1987), Current Protocols inMolecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits forconstruction of genomic libraries are also commercially available.

The cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention such as thosedisclosed herein. Probes may be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent.

The nucleic acids of interest can also be amplified from nucleic acidsamples using amplification techniques. For instance, polymerase chainreaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing or for other purposes. The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

PCR-based screening methods have been described. Wilfinger, et al.,describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study.BioTechniques, 22(3):481-486 (1997). Such methods are particularlyeffective in combination with a full-length cDNA constructionmethodology, above.

B. Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., (1979) Meth. Enzymol. 68: 90-99; thephosphodiester method of Brown, et al., (1979) Meth. Enzymol.68:109-151; the diethylphosphoramidite method of Beaucage, et al.,(1981) Tetra. Lett. 22:1859-1862; the solid phase phosphoramiditetriester method described by Beaucage and Caruthers, (1981) Tetra.Letts. 22(20):1859-1862, e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter, et al., (1984) Nucleic Acids Res.,12:6159-6168 and the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis generally produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is best employed for sequences of about 100bases or less, longer sequences may be obtained by the ligation ofshorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polypeptide of the present invention,for example a cDNA or a genomic sequence encoding a full lengthpolypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site and/ora polyadenylation signal.

A plant promoter fragment can be employed which will direct expressionof a polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoterand the GRP1-8 promoter.

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions or the presenceof light. Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress and the PPDK promoter which is inducible bylight.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds or flowers. Exemplary promoters includethe anther-specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051), glb-1 promoter and gamma-zein promoter. Also see, forexample, U.S. Patent Application Ser. Nos. 60/155,859 and 60/163,114.The operation of a promoter may also vary depending on its location inthe genome. Thus, an inducible promoter may become fully or partiallyconstitutive in certain locations.

Both heterologous and non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in recombinantexpression cassettes to drive expression of antisense nucleic acids toreduce, increase or alter concentration and/or composition of theproteins of the present invention in a desired tissue. Thus, in someembodiments, the nucleic acid construct will comprise a promoter,functional in a plant cell, operably linked to a polynucleotide of thepresent invention. Promoters useful in these embodiments include theendogenous promoters driving expression of a polypeptide of the presentinvention.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al.,PCT/US93/03868) or isolated promoters can be introduced into a plantcell in the proper orientation and distance from a cognate gene of apolynucleotide of the present invention so as to control the expressionof the gene. Gene expression can be modulated under conditions suitablefor plant growth so as to alter the total concentration and/or alter thecomposition of the polypeptides of the present invention in plant cell.Thus, the present invention provides compositions, and methods formaking, heterologous promoters and/or enhancers operably linked to anative, endogenous (i.e., non-heterologous) form of a polynucleotide ofthe present invention.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes or alternatively from another plantgene or less preferably from any other eukaryotic gene.

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusionon of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold. Buchman and Berg, (1988)Mol. Cell. Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev.1:11831200. Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofmaize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are knownin the art. See generally, The Maize Handbook, Chapter 116, Freeling andWalbot, Eds., Springer, New York (1994). The vector comprising thesequences from a polynucleotide of the present invention will typicallycomprise a marker gene which confers a selectable phenotype on plantcells. Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers, et al., (1987) Meth. in Enzymol. 153:253-277.

A polynucleotide of the present invention can be expressed in eithersense or anti-sense orientation as desired. It will be appreciated thatcontrol of gene expression in either sense or anti-sense orientation canhave a direct impact on the observable plant characteristics. Antisensetechnology can be conveniently used to inhibit gene expression inplants. To accomplish this, a nucleic acid segment from the desired geneis cloned and operably linked to a promoter such that the anti-sensestrand of RNA will be transcribed. The construct is then transformedinto plants and the antisense strand of RNA is produced. In plant cells,it has been shown that antisense RNA inhibits gene expression bypreventing the accumulation of mRNA which encodes the enzyme ofinterest, see, e.g., Sheehy, et al., (1988) Proc. Nat'l. Acad. Sci.(USA) 85:8805-8809 and Hiatt, et al., U.S. Pat. No. 4,801,340.

Another method of suppression is sense suppression (i.e.,co-supression). Introduction of nucleic acid configured in the senseorientation has been shown to be an effective means by which to blockthe transcription of target genes. For an example of the use of thismethod to modulate expression of endogenous genes see, Napoli, et al.,(1990) The Plant Cell 2:279-289 and U.S. Pat. No. 5,034,323.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff, et al., (1988)Nature 334:585-591.

A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect and/or cleave nucleicacids. For example, Vlassov, et al., (1986) Nucleic Acids Res14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, et al., (1985) Biochimie 67:785-789. Iverson and Dervan alsoshowed sequence-specific cleavage of single-stranded DNA mediated byincorporation of a modified nucleotide which was capable of activatingcleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, et al., (1989) JAm Chem Soc 111:8517-8519, effect covalent crosslinking to a targetnucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee, et al., (1988) Biochemistry 27:3197-3203. Use ofcrosslinking in triple-helix forming probes was also disclosed by Home,et al., (1990) J Am Chem Soc 112:2435-2437. Use of N4,N4-ethanocytosineas an alkylating agent to crosslink to single-stranded oligonucleotideshas also been described by Webb and Matteucci, (1986) J Am Chem Soc108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz, et al.,(1991) J. Am. Chem. Soc. 113:4000. Various compounds to bind, detect,label, and/or cleave nucleic acids are known in the art. See, forexample, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648 and5,681941.

Proteins

The isolated proteins of the present invention comprise a polypeptidehaving at least 10 amino acids from a polypeptide of the presentinvention (or conservative variants thereof) such as those encoded byany one of the polynucleotides of the present invention as discussedmore fully above (e.g., Table 1). The proteins of the present inventionor variants thereof can comprise any number of contiguous amino acidresidues from a polypeptide of the present invention, wherein thatnumber is selected from the group of integers consisting of from 10 tothe number of residues in a full-length polypeptide of the presentinvention. Optionally, this subsequence of contiguous amino acids is atleast 15, 20, 25, 30, 35 or 40 amino acids in length, often at least 50,60, 70, 80 or 90 amino acids in length. Further, the number of suchsubsequences can be any integer selected from the group consisting offrom 1 to 20, such as 2, 3, 4 or 5.

The present invention further provides a protein comprising apolypeptide having a specified sequence identity/similarity with apolypeptide of the present invention. The percentage of sequenceidentity/similarity is an integer selected from the group consisting offrom 50 to 99. Exemplary sequence identity/similarity values include55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%. Sequence identity can bedetermined using, for example, the GAP, CLUSTALW or BLAST algorithms.

As those of skill will appreciate, the present invention includes, butis not limited to, catalytically active polypeptides of the presentinvention (i.e., enzymes). Catalytically active polypeptides have aspecific activity of at least 20%, 30% or 40% and preferably at least50%, 60% or 70% and most preferably at least 80%, 90% or 95% that of thenative (non-synthetic), endogenous polypeptide. Further, the substratespecificity (k_(cat)/K_(m)) is optionally substantially similar to thenative (non-synthetic), endogenous polypeptide. Typically, the K_(m)will be at least 30%, 40%, or 50%, that of the native (non-synthetic),endogenous polypeptide; and more preferably at least 60%, 70%, 80% or90%. Methods of assaying and quantifying measures of enzymatic activityand substrate specificity (k_(cat)/K_(m)) are well known to those ofskill in the art.

Generally, the proteins of the present invention will, when presented asan immunogen, elicit production of an antibody specifically reactive toa polypeptide of the present invention. Further, the proteins of thepresent invention will not bind to antisera raised against a polypeptideof the present invention which has been fully immunosorbed with the samepolypeptide. Immunoassays for determining binding are well known tothose of skill in the art. A preferred immunoassay is a competitiveimmunoassay. Thus, the proteins of the present invention can be employedas immunogens for constructing antibodies immunoreactive to a protein ofthe present invention for such exemplary utilities as immunoassays orprotein purification techniques.

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or regulatable), followed by incorporation into anexpression vector. The vectors can be suitable for replication andintegration in either prokaryotes or eukaryotes. Typical expressionvectors contain transcription and translation terminators, initiationsequences and promoters useful for regulation of the expression of theDNA encoding a protein of the present invention. To obtain high levelexpression of a cloned gene, it is desirable to construct expressionvectors which contain, at the minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation anda transcription/translation terminator. One of skill would recognizethat modifications can be made to a protein of the present inventionwithout diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, amethionine added at the amino terminus to provide an initiation site oradditional amino acids (e.g., poly His) placed on either terminus tocreate conveniently located purification sequences. Restriction sites ortermination codons can also be introduced.

Synthesis of Proteins

The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield, et al., (1963) J. Am. Chem. Soc.85:2149-2156 and Stewart, et al., Solid Phase Peptide Synthesis, 2nded., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater lengthmay be synthesized by condensation of the amino and carboxy termini ofshorter fragments. Methods of forming peptide bonds by activation of acarboxy terminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

Purification of Proteins

The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods and others. See, for instance, Scopes,Protein Purification Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. The protein may then be isolatedfrom cells expressing the protein and further purified by standardprotein chemistry techniques as described herein. Detection of theexpressed protein is achieved by methods known in the art and include,for example, radioimmunoassays, Western blotting techniques orimmunoprecipitation.

Introduction of Nucleic Acids Into Host Cells

The method of introducing a nucleic acid of the present invention into ahost cell is not critical to the instant invention. Transformation ortransfection methods are conveniently used. Accordingly, a wide varietyof methods have been developed to insert a DNA sequence into the genomeof a host cell to obtain the transcription and/or translation of thesequence to effect phenotypic changes in the organism. Thus, any methodwhich provides for effective introduction of a nucleic acid may beemployed.

A. Plant Transformation

A nucleic acid comprising a polynucleotide of the present invention isoptionally introduced into a plant. Generally, the polynucleotide willfirst be incorporated into a recombinant expression cassette or vector.Isolated nucleic acid acids of the present invention can be introducedinto plants according to techniques known in the art. Techniques fortransforming a wide variety of higher plant species are well known anddescribed in the technical, scientific, and patent literature. See, forexample, Weising, et al., (1988) Ann. Rev. Genet. 22:421-477. Forexample, the DNA construct may be introduced directly into the genomicDNA of the plant cell using techniques such as electroporation,polyethylene glycol (PEG) poration, particle bombardment, silicon fiberdelivery or microinjection of plant cell protoplasts or embryogeniccallus. See, e.g., Tomes, et al., Direct DNA Transfer into Intact PlantCells Via Microprojectile Bombardment. pp. 197-213 in Plant Cell, Tissueand Organ Culture, Fundamental Methods. eds. Gamborg and Phillips.Springer-Verlag Berlin Heidelberg New York, 1995; see, U.S. Pat. No.5,990,387. The introduction of DNA constructs using PEG precipitation isdescribed in Paszkowski, et al., (1984) Embo J. 3:2717-2722.Electroporation techniques are described in Fromm, et al., (1985) Proc.Natl. Acad. Sci. (USA) 82:5824. Ballistic transformation techniques aredescribed in Klein, et al., (1987) Nature 327:70-73.

Agrobacterium tumefaciens-mediated transformation techniques are welldescribed in the scientific literature. See, for example, Horsch, etal., (1984) Science 233:496-498; Fraley, et al., (1983) Proc. Natl.Acad. Sci. (USA) 80:4803 and Plant Molecular Biology: A LaboratoryManual, Chapter 8, Clark, Ed., Springer-Verlag, Berlin (1997). The DNAconstructs may be combined with suitable T-DNA flanking regions andintroduced into a conventional Agrobacterium tumefaciens host vector.The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of the construct and adjacent marker into the plantcell DNA when the cell is infected by the bacteria. See, U.S. Pat. No.5,591,616. Although Agrobacterium is useful primarily in dicots, certainmonocots can be transformed by Agrobacterium. For instance,Agrobacterium transformation of maize is described in U.S. Pat. No.5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, Rigby, Ed.,London, Academic Press, 1987; and Lichtenstein, and Draper, In: DNACloning, Vol. II, Glover, Ed., Oxford, IRI Press, 1985), PCT ApplicationNumber PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) describes theuse of A. rhizogenes strain A4 and its Ri plasmid along with A.tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA uptake(see, e.g., Freeman, et al., (1984) Plant Cell Physiol. 25:1353), (3)the vortexing method (see, e.g., Kindle, (1990) Proc. Natl. Acad. Sci.,(USA) 87:1228).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou, et al., (1983) Methods in Enzymology101:433; Hess, (1987) Intern Rev. Cytol. 107:367; Luo, et al., (1988)Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena, et al., (1987) Nature, 325.274. DNA can alsobe injected directly into the cells of immature embryos and therehydration of desiccated embryos as described by Neuhaus, et al.,(1987) Theor. Appl. Genet., 75:30 and Benbrook, et al., in ProceedingsBio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A varietyof plant viruses that can be employed as vectors are known in the artand include cauliflower mosaic virus (CaMV), geminivirus, brome mosaicvirus, and tobacco mosaic virus.

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,biolistics and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler,Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson andRoss, Inc. (1977).

Transgenic Plant Regeneration

Plant cells which directly result or are derived from the nucleic acidintroduction techniques can be cultured to regenerate a whole plantwhich possesses the introduced genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium. Plants cells can be regenerated, e.g., from single cells,callus tissue or leaf discs according to standard plant tissue culturetechniques. It is well known in the art that various cells, tissues, andorgans from almost any plant can be successfully cultured to regeneratean entire plant. Plant regeneration from cultured protoplasts isdescribed in Evans, et al., Protoplasts Isolation and Culture, Handbookof Plant Cell Culture, Macmillan Publishing Company, New York, pp.124-176 (1983) and Binding, Regeneration of Plants, Plant Protoplasts,CRC Press, Boca Raton, pp. 21-73 (1985).

The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, Weissbach and Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting the transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, New York (1994); Corn and CornImprovement, 3^(rd) edition, Sprague and Dudley Eds., American Societyof Agronomy, Madison, Wis. (1988). For transformation and regenerationof maize see, Gordon-Kamm, et al., (1990) The Plant Cell 2:603-618.

The regeneration of plants containing the polynucleotide of the presentinvention and introduced by Agrobacterium from leaf explants can beachieved as described by Horsch, et al., (1985) Science, 227:1229-1231.In this procedure, transformants are grown in the presence of aselection agent and in a medium that induces the regeneration of shootsin the plant species being transformed as described by Fraley, et al.,(1983) Proc. Natl. Acad. Sci. (U.S.A.) 80:4803. This procedure typicallyproduces shoots within two to four weeks and these transformant shootsare then transferred to an appropriate root-inducing medium containingthe selective agent and an antibiotic to prevent bacterial growth.Transgenic plants of the present invention may be fertile or sterile.

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self-crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype. Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences.

Transgenic plants expressing a polynucleotide of the present inventioncan be screened for transmission of the nucleic acid of the presentinvention by, for example, standard immunoblot and DNA detectiontechniques. Expression at the RNA level can be determined initially toidentify and quantitate expression-positive plants. Standard techniquesfor RNA analysis can be employed and include PCR amplification assaysusing oligonucleotide primers designed to amplify only the heterologousRNA templates and solution hybridization assays using heterologousnucleic acid-specific probes. The RNA-positive plants can then analyzedfor protein expression by Western immunoblot analysis using thespecifically reactive antibodies of the present invention. In addition,in situ hybridization and immunocytochemistry according to standardprotocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

Modulating Polypeptide Levels and/or Composition

The present invention further provides a method for modulating (i.e.,increasing or decreasing) the concentration or ratio of the polypeptidesof the present invention in a plant or part thereof. Modulation can beeffected by increasing or decreasing the concentration and/or the ratioof the polypeptides of the present invention in a plant. The methodcomprises introducing into a plant cell a recombinant expressioncassette comprising a polynucleotide of the present invention asdescribed above to obtain a transgenic plant cell, culturing thetransgenic plant cell under transgenic plant cell growing conditions andinducing or repressing expression of a polynucleotide of the presentinvention in the transgenic plant for a time sufficient to modulateconcentration and/or the ratios of the polypeptides in the transgenicplant or plant part.

In some embodiments, the concentration and/or ratios of polypeptides ofthe present invention in a plant may be modulated by altering, in vivoor in vitro, the promoter of a gene to up- or down-regulate geneexpression. In some embodiments, the coding regions of native genes ofthe present invention can be altered via substitution, addition,insertion or deletion to decrease activity of the encoded enzyme. (See,e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868.)And in some embodiments, an isolated nucleic acid (e.g., a vector)comprising a promoter sequence is transfected into a plant cell.Subsequently, a plant cell comprising the promoter operably linked to apolynucleotide of the present invention is selected for by means knownto those of skill in the art such as, but not limited to, Southern blot,DNA sequencing or PCR analysis using primers specific to the promoterand to the gene and detecting amplicons produced therefrom. A plant orplant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate theconcentration and/or ratios of polypeptides of the present invention inthe plant. Plant forming conditions are well known in the art anddiscussed briefly, supra.

In general, concentration or the ratios of the polypeptides is increasedor decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or90% relative to a native control plant, plant part, or cell lacking theaforementioned recombinant expression cassette. Modulation in thepresent invention may occur during and/or subsequent to growth of theplant to the desired stage of development. Modulating nucleic acidexpression temporally and/or in particular tissues can be controlled byemploying the appropriate promoter operably linked to a polynucleotideof the present invention in, for example, sense or antisense orientationas discussed in greater detail, supra. Induction of expression of apolynucleotide of the present invention can also be controlled byexogenous administration of an effective amount of inducing compound.Inducible promoters and inducing compounds which activate expressionfrom these promoters are well known in the art. In preferredembodiments, the polypeptides of the present invention are modulated inmonocots, particularly maize.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, (1987) Nucleic Acids Res.15:8125) and the 7-methylguanosine cap structure (Drummond, et al.,(1985) Nucleic Acids Res. 13:7375). Negative elements include stableintramolecular 5′ UTR stem-loop structures (Muesing, et al., (1987) Cell48:691) and AUG sequences or short open reading frames preceded by anappropriate AUG in the 5′ UTR (Kozak, supra, Rao, et al., (1988) Mol.and Cell. Biol. 8:284). Accordingly, the present invention provides 5′and/or 3′ untranslated regions for modulation of translation ofheterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host such as tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see, Devereaux, etal., (1984) Nucleic Acids Res. 12:387-395) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5, 10, 20, 50 or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingtherefrom. Sequence shuffling is described in PCT Publication Number WO97/20078. See also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509. Generally, sequence shuffling provides a means forgenerating libraries of polynucleotides having a desired characteristicwhich can be selected or screened for. Libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides which comprise sequence regions which have substantialsequence identity and can be homologously recombined in vitro or invivo. The population of sequence-recombined polynucleotides comprises asubpopulation of polynucleotides which possess desired or advantageouscharacteristics and which can be selected by a suitable selection orscreening method. The characteristics can be any property or attributecapable of being selected for or detected in a screening system and mayinclude properties of: an encoded protein, a transcriptional element, asequence controlling transcription, RNA processing, RNA stability,chromatin conformation, translation, or other expression property of agene or transgene, a replicative element, a protein-binding element orthe like, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be adecreased K_(m) and/or increased K_(cat) over the wild-type protein asprovided herein. In other embodiments, a protein or polynucleotidegenerated from sequence shuffling will have a ligand binding affinitygreater than the non-shuffled wild-type polynucleotide. The increase insuch properties can be at least 110%, 120%, 130%, 140% or at least 150%of the wild-type value.

Generic and Consensus Sequences

Polynucleotides and polypeptides of the present invention furtherinclude those having: (a) a generic sequence of at least two homologouspolynucleotides or polypeptides, respectively, of the present inventionand (b) a consensus sequence of at least three homologouspolynucleotides or polypeptides, respectively, of the present invention.The generic sequence of the present invention comprises each species ofpolypeptide or polynucleotide embraced by the generic polypeptide orpolynucleotide sequence, respectively. The individual speciesencompassed by a polynucleotide having an amino acid or nucleic acidconsensus sequence can be used to generate antibodies or produce nucleicacid probes or primers to screen for homologs in other species, genera,families, orders, classes, phyla or kingdoms. For example, apolynucleotide having a consensus sequence from a gene family of Zeamays can be used to generate antibody or nucleic acid probes or primersto other Gramineae species such as wheat, rice or sorghum.Alternatively, a polynucleotide having a consensus sequence generatedfrom orthologous genes can be used to identify or isolate orthologs ofother taxa. Typically, a polynucleotide having a consensus sequence willbe at least 9, 10, 15, 20, 25, 30 or 40 amino acids in length, or 20,30, 40, 50, 100 or 150 nucleotides in length. As those of skill in theart are aware, a conservative amino acid substitution can be used foramino acids which differ amongst aligned sequence but are from the sameconservative substitution group as discussed above. Optionally, no morethan 1 or 2 conservative amino acids are substituted for each 10 aminoacid length of consensus sequence.

Similar sequences used for generation of a consensus or generic sequenceinclude any number and combination of allelic variants of the same gene,orthologous or paralogous sequences as provided herein. Optionally,similar sequences used in generating a consensus or generic sequence areidentified using the BLAST algorithm's smallest sum probability (P(N)).Various suppliers of sequence-analysis software are listed in chapter 7of Current Protocols in Molecular Biology, Ausubel et al., Eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (Supplement 30). A polynucleotide sequenceis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, or 0.001 and most preferably less than about 0.0001 or 0.00001.Similar polynucleotides can be aligned and a consensus or genericsequence generated using multiple sequence alignment software availablefrom a number of commercial suppliers such as the Genetics ComputerGroup's (Madison, Wis.) PILEUP software, Vector NTI's (North Bethesda,Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.) SEQUENCHER. Conveniently,default parameters of such software can be used to generate consensus orgeneric sequences.

Machine Applications

The present invention provides machines, data structures, and processesfor modeling or analyzing the polynucleotides and polypeptides of thepresent invention.

A. Machines: Data, Data Structures, Processes and Functions

The present invention provides a machine having a memory comprising: 1)data representing a sequence of a polynucleotide or polypeptide of thepresent invention, 2) a data structure which reflects the underlyingorganization and structure of the data and facilitates program access todata elements corresponding to logical sub-components of the sequence,3) processes for effecting the use, analysis, or modeling of thesequence, and 4) optionally, a function or utility for thepolynucleotide or polypeptide. Thus, the present invention provides amemory for storing data that can be accessed by a computer programmed toimplement a process for affecting the use, analyses or modeling of asequence of a polynucleotide, with the memory comprising datarepresenting the sequence of a polynucleotide of the present invention.

The machine of the present invention is typically a digital computer.The term “computer” includes one or several desktop or portablecomputers, computer workstations, servers (including intranet orinternet servers), mainframes and any integrated system comprising anyof the above irrespective of whether the processing, memory, input oroutput of the computer is remote or local, as well as any networkinginterconnecting the modules of the computer. The term “computer” isexclusive of computers of the United States Patent and Trademark Officeor the European Patent Office when data representing the sequence ofpolypeptides or polynucleotides of the present invention is used forpatentability searches.

The present invention contemplates providing as data a sequence of apolynucleotide of the present invention embodied in a computer readablemedium. As those of skill in the art will be aware, the form of memoryof a machine of the present invention or the particular embodiment ofthe computer readable medium, are not critical elements of the inventionand can take a variety of forms. The memory of such a machine includes,but is not limited to, ROM or RAM or computer readable media such as,but not limited to, magnetic media such as computer disks or hard drivesor media such as CD-ROMs, DVDs and the like.

The present invention further contemplates providing a data structurethat is also contained in memory. The data structure may be defined bythe computer programs that define the processes (see below) or it may bedefined by the programming of separate data storage and retrievalprograms subroutines or systems. Thus, the present invention provides amemory for storing a data structure that can be accessed by a computerprogrammed to implement a process for affecting the use, analysis ormodeling of a sequence of a polynucleotide. The memory comprises datarepresenting a polynucleotide having the sequence of a polynucleotide ofthe present invention. The data is stored within memory. Further, a datastructure, stored within memory, is associated with the data reflectingthe underlying organization and structure of the data to facilitateprogram access to data elements corresponding to logical sub-componentsof the sequence. The data structure enables the polynucleotide to beidentified and manipulated by such programs.

In a further embodiment, the present invention provides a data structurethat contains data representing a sequence of a polynucleotide of thepresent invention stored within a computer readable medium. The datastructure is organized to reflect the logical structuring of thesequence, so that the sequence is easily analyzed by software programscapable of accessing the data structure. In particular, the datastructures of the present invention organize the reference sequences ofthe present invention in a manner which allows software tools to performa wide variety of analyses using logical elements and sub-elements ofeach sequence.

An example of such a data structure resembles a layered hash table,where in one dimension the base content of the sequence is representedby a string of elements A, T, C, G and N. The direction from the 5′ endto the 3′ end is reflected by the order from the position 0 to theposition of the length of the string minus one. Such a string,corresponding to a nucleotide sequence of interest, has a certain numberof substrings, each of which is delimited by the string position of its5′ end and the string position of its 3′ end within the parent string.In a second dimension, each substring is associated with or pointed toone or multiple attribute fields. Such attribute fields containannotations to the region on the nucleotide sequence represented by thesubstring.

For example, a sequence under investigation is 520 bases long andrepresented by a string named SeqTarget. There is a minor groove in the5′ upstream non-coding region from position 12 to 38, which isidentified as a binding site for an enhancer protein HM-A, which in turnwill increase the transcription of the gene represented by SeqTarget.Here, the substring is represented as (12, 38) and has the followingattributes: [upstream uncoded], [minor groove], [HM-A binding] and[increase transcription upon binding by HM-A]. Similarly, other types ofinformation can be stored and structured in this manner, such asinformation related to the whole sequence, e.g., whether the sequence isa full length viral gene, a mammalian house keeping gene or an EST fromclone X, information related to the 3′ down stream non-coding region,e.g., hair pin structure and information related to various domains ofthe coding region, e.g., Zinc finger.

This data structure is an open structure and is robust enough toaccommodate newly generated data and acquired knowledge. Such astructure is also a flexible structure. It can be trimmed down to a 1-Dstring to facilitate data mining and analysis steps, such as clustering,repeat-masking, and HMM analysis. Meanwhile, such a data structure alsocan extend the associated attributes into multiple dimensions. Pointerscan be established among the dimensioned attributes when needed tofacilitate data management and processing in a comprehensive genomicsknowledgebase. Furthermore, such a data structure is object-oriented.Polymorphism can be represented by a family or class of sequenceobjects, each of which has an internal structure as discussed above. Thecommon traits are abstracted and assigned to the parent object, whereaseach child object represents a specific variant of the family or class.Such a data structure allows data to be efficiently retrieved, updatedand integrated by the software applications associated with the sequencedatabase and/or knowledgebase.

The present invention contemplates providing processes for effectinganalysis and modeling, which are described in the following section.

Optionally, the present invention further contemplates that the machineof the present invention will embody in some manner a utility orfunction for the polynucleotide or polypeptide of the present invention.The function or utility of the polynucleotide or polypeptide can be afunction or utility for the sequence data, per se, or of the tangiblematerial. Exemplary function or utilities include the name (perInternational Union of Biochemistry and Molecular Biology rules ofnomenclature) or function of the enzyme or protein represented by thepolynucleotide or polypeptide of the present invention; the metabolicpathway of the protein represented by the polynucleotide or polypeptideof the present invention; the substrate or product or structural role ofthe protein represented by the polynucleotide or polypeptide of thepresent invention or the phenotype (e.g., an agronomic orpharmacological trait) affected by modulating expression or activity ofthe protein represented by the polynucleotide or polypeptide of thepresent invention.

B. Computer Analysis and Modeling

The present invention provides a process of modeling and analyzing datarepresentative of a polynucleotide or polypeptide sequence of thepresent invention. The process comprises entering sequence data of apolynucleotide or polypeptide of the present invention into a machinehaving a hardware or software sequence modeling and analysis system,developing data structures to facilitate access to the sequence data,manipulating the data to model or analyze the structure or activity ofthe polynucleotide or polypeptide and displaying the results of themodeling or analysis. Thus, the present invention provides a process foraffecting the use, analysis or modeling of a polynucleotide sequence orits derived peptide sequence through use of a computer having a memory.The process comprises: 1) placing into the memory data representing apolynucleotide having the sequence of a polynucleotide of the presentinvention, developing within the memory a data structure associated withthe data and reflecting the underlying organization and structure of thedata to facilitate program access to data elements corresponding tological sub-components of the sequence, 2) programming the computer witha program containing instructions sufficient to implement the processfor effecting the use, analysis or modeling of the polynucleotidesequence or the peptide sequence and 3) executing the program on thecomputer while granting the program access to the data and to the datastructure within the memory.

A variety of modeling and analytic tools are well known in the art andavailable commercially. Included amongst the modeling/analysis tools aremethods to: 1) recognize overlapping sequences (e.g., from a sequencingproject) with a polynucleotide of the present invention and create analignment called a “contig”; 2) identify restriction enzyme sites of apolynucleotide of the present invention; 3) identify the products of aT1 ribonuclease digestion of a polynucleotide of the present invention;4) identify PCR primers with minimal self-complementarity; 5) computepairwise distances between sequences in an alignment, reconstructphylogentic trees using distance methods and calculate the degree ofdivergence of two protein coding regions; 6) identify patterns such ascoding regions, terminators, repeats and other consensus patterns inpolynucleotides of the present invention; 7) identify RNA secondarystructure; 8) identify sequence motifs, isoelectric point, secondarystructure, hydrophobicity and antigenicity in polypeptides of thepresent invention; 9) translate polynucleotides of the present inventionand backtranslate polypeptides of the present invention and 10) comparetwo protein or nucleic acid sequences and identifying points ofsimilarity or dissimilarity between them.

The processes for effecting analysis and modeling can be producedindependently or obtained from commercial suppliers. Exemplary analysisand modeling tools are provided in products such as InforMax's(Bethesda, Md.) Vector NTI Suite (Version 5.5), Intelligenetics'(Mountain View, Calif.) PC/Gene program and Genetics Computer Group's(Madison, Wis.) Wisconsin Package® (Version 10.0); these tools, and thefunctions they perform, (as provided and disclosed by the programs andaccompanying literature) are incorporated herein by reference and aredescribed in more detail in section C which follows.

Thus, in a further embodiment, the present invention provides amachine-readable media containing a computer program and data,comprising a program stored on the media containing instructionssufficient to implement a process for affecting the use, analysis ormodeling of a representation of a polynucleotide or peptide sequence.The data stored on the media represents a sequence of a polynucleotidehaving the sequence of a polynucleotide of the present invention. Themedia also includes a data structure reflecting the underlyingorganization and structure of the data to facilitate program access todata elements corresponding to logical sub-components of the sequence,the data structure being inherent in the program and in the way in whichthe program organizes and accesses the data.

C. Homology Searches

As an example of such a comparative analysis, the present inventionprovides a process of identifying a candidate homologue (i.e., anortholog or paralog) of a polynucleotide or polypeptide of the presentinvention. The process comprises entering sequence data of apolynucleotide or polypeptide of the present invention into a machinehaving a hardware or software sequence analysis system, developing datastructures to facilitate access to the sequence data, manipulating thedata to analyze the structure the polynucleotide or polypeptide anddisplaying the results of the analysis. A candidate homologue hasstatistically significant probability of having the same biologicalfunction (e.g., catalyzes the same reaction, binds to homologousproteins/nucleic acids, has a similar structural role) as the referencesequence to which it is compared. Accordingly, the polynucleotides andpolypeptides of the present invention have utility in identifyinghomologs in animals or other plant species, particularly those in thefamily Gramineae such as, but not limited to, sorghum, wheat or rice.

The process of the present invention comprises obtaining datarepresenting a polynucleotide or polypeptide test sequence. Testsequences can be obtained from a nucleic acid of an animal or plant.Test sequences can be obtained directly or indirectly from sequencedatabases including, but not limited to, those such as: GenBank, EMBL,GenSeq, SWISS-PROT or those available on-line via the UK Human GenomeMapping Project (HGMP) GenomeWeb. In some embodiments the test sequenceis obtained from a plant species other than maize whose function isuncertain but will be compared to the test sequence to determinesequence similarity or sequence identity. The test sequence data isentered into a machine, such as a computer, containing: i) datarepresenting a reference sequence and ii) a hardware or softwaresequence comparison system to compare the reference and test sequencefor sequence similarity or identity.

Exemplary sequence comparison systems are provided for in sequenceanalysis software such as those provided by the Genetics Computer Group(Madison, Wis.) or InforMax (Bethesda, Md.) or Intelligenetics (MountainView, Calif.). Optionally, sequence comparison is established using theBLAST or GAP suite of programs. Generally, a smallest sum probabilityvalue (P(N)) of less than 0.1, or alternatively, less than 0.01, 0.001,0.0001 or 0.00001 using the BLAST 2.0 suite of algorithms under defaultparameters identifies the test sequence as a candidate homologue (i.e.,an allele, ortholog or paralog) of the reference sequence. Those ofskill in the art will recognize that a candidate homologue has anincreased statistical probability of having the same or similar functionas the gene/protein represented by the test sequence.

The reference sequence can be the sequence of a polypeptide or apolynucleotide of the present invention. The reference or test sequenceis each optionally at least 25 amino acids or at least 100 nucleotidesin length. The length of the reference or test sequences can be thelength of the polynucleotide or polypeptide described, respectively,above in the sections entitled “Nucleic Acids” (particularly section(g)) and “Proteins”. As those of skill in the art are aware, the greaterthe sequence identity/similarity between a reference sequence of knownfunction and a test sequence, the greater the probability that the testsequence will have the same or similar function as the referencesequence. The results of the comparison between the test and referencesequences are outputted (e.g., displayed, printed, recorded) via any oneof a number of output devices and/or media (e.g., computer monitor, hardcopy or computer readable medium).

Detection of Nucleic Acids

The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of containing a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of maize. In someembodiments, a cognate gene of a polynucleotide of the present inventionor portion thereof can be amplified prior to the step of contacting thenucleic acid sample with a polynucleotide of the present invention. Thenucleic acid sample is contacted with the polynucleotide to form ahybridization complex. The polynucleotide hybridizes under stringentconditions to a gene encoding a polypeptide of the present invention.Formation of the hybridization complex is used to detect a gene encodinga polypeptide of the present invention in the nucleic acid sample. Thoseof skill will appreciate that an isolated nucleic acid comprising apolynucleotide of the present invention should lack cross-hybridizingsequences in common with non-target genes that would yield a falsepositive result. Detection of the hybridization complex can be achievedusing any number of well known methods. For example, the nucleic acidsample, or a portion thereof, may be assayed by hybridization formatsincluding but not limited to, solution phase, solid phase, mixed phaseor in situ hybridization assays.

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, radioisotopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads, fluorescent dyes,radiolabels, enzymes and colorimetric labels. Other labels includeligands which bind to antibodies labeled with fluorophores,chemiluminescent agents and enzymes. Labeling the nucleic acids of thepresent invention is readily achieved such as by the use of labeled PCRprimers.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

Example 1

This example describes the construction of a cDNA library.

Total RNA can be isolated from maize tissues with TRIzol Reagent (LifeTechnology Inc. Gaithersburg, Md.) using a modification of the guanidineisothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi(Chomczynski and Sacchi, (1987) Anal. Biochem. 162:156). In brief, planttissue samples is pulverized in liquid nitrogen before the addition ofthe TRIzol Reagent and then further homogenized with a mortar andpestle. Addition of chloroform followed by centrifugation is conductedfor separation of an aqueous phase and an organic phase. The total RNAis recovered by precipitation with isopropyl alcohol from the aqueousphase.

The selection of poly(A)+ RNA from total RNA can be performed usingPolyATact system (Promega Corporation. Madison, Wis.). Biotinylatedoligo(dT) primers are used to hybridize to the 3′ poly(A) tails on mRNA.The hybrids are captured using streptavidin coupled to paramagneticparticles and a magnetic separation stand. The mRNA is then washed athigh stringency conditions and eluted by RNase-free deionized water.

cDNA synthesis and construction of unidirectional cDNA libraries can beaccomplished using the SuperScript Plasmid System (Life Technology Inc.Gaithersburg, Md.). The first strand of cDNA is synthesized by primingan oligo(dT) primer containing a Not I site. The reaction is catalyzedby SuperScript Reverse Transcriptase II at 45° C. The second strand ofcDNA is labeled with alpha-³²P-dCTP and a portion of the reactionanalyzed by agarose gel electrophoresis to determine cDNA sizes. cDNAmolecules smaller than 500 base pairs and unligated adapters are removedby Sephacryl-5400 chromatography. The selected cDNA molecules areligated into pSPORT1 vector in between of Not I and Sal I sites.

Alternatively, cDNA libraries can be prepared by any one of many methodsavailable. For example, the cDNAs may be introduced into plasmid vectorsby first preparing the cDNA libraries in Uni-ZAP™ XR vectors accordingto the manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). The Uni-ZAP™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH10B cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see, Adams, et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2

This method describes construction of a full-length enriched cDNAlibrary.

An enriched full-length cDNA library can be constructed using one of twovariations of the method of Carninci, et al., (1996) Genomics37:327-336. These variations are based on chemical introduction of abiotin group into the diol residue of the 5′ cap structure of eukaryoticmRNA to select full-length first strand cDNA. The selection occurs bytrapping the biotin residue at the cap sites using streptavidin-coatedmagnetic beads followed by RNase I treatment to eliminate incompletelysynthesized cDNAs. Second strand cDNA is synthesized using establishedprocedures such as those provided in Life Technologies' (Rockville, Md.)“SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning” kit.Libraries made by this method have been shown to contain 50% to 70%full-length cDNAs.

The first strand synthesis methods are detailed below. An asteriskdenotes that the reagent was obtained from Life Technologies, Inc.

A. First Strand cDNA Synthesis Method 1 (with Trehalose)

mRNA (10 ug) 25 μl *Not I primer (5 ug) 10 μl *5× 1^(st) strand buffer43 μl *0.1m DTT 20 μl *dNTP mix 10 mm 10 μl BSA 10 ug/μl  1 μl Trehalose(saturated) 59.2 μl   RNase inhibitor (Promega) 1.8 μl  *Superscript IIRT 200 u/μl 20 μl 100% glycerol 18 μl Water  7 μl

The mRNA and Not I primer are mixed and denatured at 65° C. for 10 min.They are then chilled on ice and other components added to the tube.Incubation is at 45° C. for 2 min. Twenty microliters of RT (reversetranscriptase) is added to the reaction and start program on thethermocycler (MJ Research, Waltham, Mass.):

Step 1 45° C. 10 min Step 2 45° C. −0.3° C./cycle, 2 seconds/cycle Step3 go to 2 for 33 cycles Step 4 35° C. 5 min Step 5 45° C. 5 min Step 645° C. 0.2° C./cycle, 1 sec/cycle Step 7 go to 7 for 49 cycles Step 855° C. 0.1° C./cycle, 12 sec/cycle Step 9 go to 8 for 49 cycles Step 1055° C. 2 min Step 11 60° C. 2 min Step 12 go to 11 for 9 times Step 13 4° C. forever Step 14 endB. First Strand cDNA Synthesis Method 2

mRNA (10 μg) 25 μl water 30 μl *Not I adapter primer (5 μg) 10 μl 65° C.for 10 min, chill on ice, then add following reagents, *5× first buffer20 μl *0.1M DTT 10 μl *10 mM dNTP mix  5 μl

Incubate at 45° C. for 2 min, then add 10 μl of *Superscript II RT (200u/μl), start the following program:

Step 1 45° C. for 6 sec, −0.1° C./cycle Step 2 go to 1 for 99 additionalcycles Step 3 35° C. for 5 min Step 4 45° C. for 60 min Step 5 50° C.for 10 min Step 6  4° C. forever Step 7 end

After the 1^(st) strand cDNA synthesis, the DNA is extracted by phenolaccording to standard procedures, and then precipitated in NaOAc andethanol, and stored in −20° C.

C. Oxidization of the Diol Group of mRNA for Biotin Labeling

First strand cDNA is spun down and washed once with 70% EtOH. The pelletresuspended in 23.2 μl of DEPC treated water and put on ice. Prepare 100mM of NaIO4 freshly, and then add the following reagents:

mRNA: 1^(st) cDNA (start with 20 μg mRNA) 46.4 μl  100 mM NaIO4 (freshlymade) 2.5 μl NaOAc 3M pH 4.5 1.1 μl

To make 100 mM NaIO4, use 21.39 μg of NaIO4 for 1 μl of water.

Wrap the tube in a foil and incubate on ice for 45 min.

After the incubation, the reaction is then precipitated in:

5M NaCl 10 μl 20% SDS 0.5 μl  isopropanol 61 μl

Incubate on ice for at least 30 min, then spin it down at max speed at4° C. for 30 min and wash once with 70% ethanol and then 80% EtOH.

D. Biotinylation of the mRNA Diol Group

Resuspend the DNA in 110 μl DEPC treated water, then add the followingreagents:

20% SDS 5 μl 2M NaOAc pH 6.1 5 μl 10 mm biotin hydrazide (freshly made)300 μl 

Wrap in a foil and incubate at room temperature overnight.

E. RNase I Treatment

Precipitate DNA in:

5M NaCl 10 μl 2M NaOAc pH 6.1 75 μl biotinylated mRNA:cDNA 420 μl  100%EtOH (2.5Vol) 1262.5 μl   

(Perform this precipitation in two tubes and split the 420 μl of DNAinto 210 μl each, add 5 μl of 5M NaCl, 37.5 μl of 2M NaOAc pH 6.1 and631.25 μl of 100% EtOH).

Store at −20° C. for at least 30 min. Spin the DNA down at 4° C. atmaximal speed for 30 min. and wash with 80% EtOH twice, then dissolveDNA in 70 μl RNase free water. Pool two tubes and end up with 140 μl.

Add the following reagents:

RNase One 10 U/μl 40 μl 1^(st) cDNA:RNA 140 μl  10× buffer 20 μl

Incubate at 37° C. for 15 min.

Add 5 μl of 40 μg/μl yeast tRNA to each sample for capturing.

F. Full length 1^(st) cDNA capturing

Blocking the beads with yeast tRNA:

Beads 1 ml Yeast tRNA 40 μg/μl 5 μl

Incubate on ice for 30 min with mixing, wash 3 times with 1 ml of 2MNaCl, 50 mmEDTA, pH 8.0.

Resuspend the beads in 800 μl of 2M NaCl, 50 mm EDTA, pH 8.0, add RNaseI treated sample 200 μl, and incubate the reaction for 30 min at roomtemperature.

Capture the beads using the magnetic stand, save the supernatant, andstart following washes:

2 washes with 2M NaCl, 50 mm EDTA, pH 8.0, 1 ml each time,

1 wash with 0.4% SDS, 50 μg/ml tRNA,

1 wash with 10 mm Tris-Cl pH 7.5, 0.2 mm EDTA, 10 mm NaCl, 20% glycerol,

1 wash with 50 μg/ml tRNA, 1 wash with 1^(st) cDNA buffer

G. Second strand cDNA synthesis

Resuspend the beads in:

*5× first buffer 8 μl *0.1 mM DTT 4 μl *10 mm dNTP mix 8 μl *5× 2ndbuffer 60 μl  *E. coli Ligase 10 U/μl 2 μl *E. coli DNA polymerase 10U/μl 8 μl *E. coli RNaseH 2 U/μl 2 μl P32 dCTP 10 μci/μl 2 μl Or waterup to 300 μl 208 μl 

Incubate at 16° C. for 2 hr with mixing the reaction in every 30 min.

Add 4 μl of T4 DNA polymerase and incubate for additional 5 min at 16°C.

Elute 2^(nd) cDNA from the beads.

Use a magnetic stand to separate the 2^(nd) cDNA from the beads, thenresuspend the beads in 200 μl of water, and then separate again, poolthe samples (about 500 μl),

Add 200 μl of water to the beads, then 200 μl of phenol:chloroform,vortex and spin to separate the sample with phenol.

Pool the DNA together (about 700 μl) and use phenol to clean the DNAagain, DNA is then precipitated in 2 μg of glycogen and 0.5 vol of 7.5MNH4OAc and 2 vol of 100% EtOH. Precipitate overnight. Spin down thepellet and wash with 70% EtOH, air-dry the pellet.

DNA 250 μl DNA 200 μl 7.5M NH4OAc 125 μl 7.5M NH4OAc 100 μl 100% EtOH750 μl 100% EtOH 600 μl glycogen 1 μg/μl  2 μl glycogen 1 μg/μl  2 μlH. Sal I Adapter Ligation

Resuspend the pellet in 26 μl of water and use 1 μl for TAE gel.

Set up reaction as following:

2^(nd) strand cDNA 25 μl *5× T4 DNA ligase buffer 10 μl *Sal I adapters10 μl *T4 DNA ligase  5 μl

Mix gently, incubate the reaction at 16° C. overnight.

Add 2 μl of ligase second day and incubate at room temperature for 2 hrs(optional).

Add 50 μl water to the reaction and use 100 μl of phenol to clean theDNA, 90 μl of the upper phase is transferred into a new tube andprecipitate in:

Glycogen 1 μg/μl  2 μl Upper phase DNA 90 μl 7.5M NH4OAc 50 μl 100% EtOH300 μl precipitate at −20° C. overnight

Spin down the pellet at 4° C. and wash in 70% EtOH, dry the pellet.

I. Not I Digestion

2^(nd) cDNA 41 μl  *Reaction 3 buffer 5 μl *Not I 15 u/μl 4 μl

Mix gently and incubate the reaction at 37° C. for 2 hr.

Add 50 μl of water and 100 μl of phenol, vortex, and take 90 μl of theupper phase to a new tube, then add 50 μl of NH₄OAc and 300 μl of EtOH.Precipitate overnight at −20° C.

Cloning, ligation and transformation are performed per the SuperscriptcDNA synthesis kit.

Example 3

This example describes cDNA sequencing and library subtraction.

Individual colonies can be picked and DNA prepared either by PCR withM13 forward primers and M13 reverse primers or by plasmid isolation.cDNA clones can be sequenced using M13 reverse primers.

cDNA libraries are plated out on 22×22 cm² agar plate at density ofabout 3,000 colonies per plate. The plates are incubated in a 37° C.incubator for 12-24 hours. Colonies are picked into 384-well plates by arobot colony picker, Q-bot (GENETIX Limited). These plates are incubatedovernight at 37° C. Once sufficient colonies are picked, they are pinnedonto 22×22 cm² nylon membranes using Q-bot. Each membrane holds 9,216 or36,864 colonies. These membranes are placed onto an agar plate with anappropriate antibiotic. The plates are incubated at 37° C. overnight.

After colonies are recovered on the second day, these filters are placedon filter paper prewetted with denaturing solution for four minutes,then incubated on top of a boiling water bath for an additional fourminutes. The filters are then placed on filter paper prewetted withneutralizing solution for four minutes. After excess solution is removedby placing the filters on dry filter papers for one minute, the colonyside of the filters is placed into Proteinase K solution, incubated at37° C. for 40-50 minutes. The filters are placed on dry filter papers todry overnight. DNA is then cross-linked to nylon membrane by UV lighttreatment

Colony hybridization is conducted as described by Sambrook, et al., (inMolecular Cloning: A laboratory Manual, 2^(nd) Edition). The followingprobes can be used in colony hybridization:

-   -   1. First strand cDNA from the same tissue as the library was        made from to remove the most redundant clones.    -   2. 48-192 most redundant cDNA clones from the same library based        on previous sequencing data.    -   3. 192 most redundant cDNA clones in the entire maize sequence        database.    -   4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAA        AAA AAA AAA AAA, SEQ ID NO: 31, removes clones containing a poly        A tail but no cDNA.    -   5. cDNA clones derived from rRNA.

The image of the autoradiography is scanned into computer and the signalintensity and cold colony addresses of each colony is analyzed.Re-arraying of cold-colonies from 384 well plates to 96 well plates isconducted using Q-bot.

Example 4

This example describes identification of the gene from a computerhomology search.

Gene identities can be determined by conducting BLAST (Basic LocalAlignment Search Tool; Altschul, et al., (1993) J. Mol. Biol.215:403-410) searches under default parameters for similarity tosequences contained in the BLAST “nr” database (comprising allnon-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL and DDBJdatabases). The cDNA sequences are analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm. The DNA sequences are translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States, (1993) Nature Genetics 3:266-272) provided by theNCBI. In some cases, the sequencing data from two or more clonescontaining overlapping segments of DNA are used to construct contiguousDNA sequences.

Sequence alignments and percent identity calculations can be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences can be performed using the Clustal method of alignment(Higgins and Sharp, (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method are KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

Other methods of sequence alignment and percent identity analysis knownto those of skill in the art, including those disclosed herein, can alsobe employed.

Example 5

This example describes expression of transgenes in monocot cells.

A transgene comprising a cDNA encoding the instant polypeptides in senseorientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209)and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (SequenaseDNA Sequencing Kit; US Biochemical). The resulting plasmid constructwould comprise a transgene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides and the 10 kD zein 3′ region.

The transgene described above can then be introduced into maize cells bythe following procedure. Immature maize embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred maize lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu, et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are keptin the dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (Hoechst Ag, Frankfurt, Germany) or equivalent maybe used in transformation experiments in order to provide for aselectable marker. This plasmid contains the Pat gene (see, EP PatentPublication Number 0 242 236) which encodes phosphinothricin acetyltransferase (PAT). The enzyme PAT confers resistance to herbicidalglutamine synthetase inhibitors such as phosphinothricin. The pat genein p35S/Ac is under the control of the 35S promoter from CauliflowerMosaic Virus (Odell, et al., (1985) Nature 313:810-812) and the 3′region of the nopaline synthase gene from the T-DNA of the Ti plasmid ofAgrobacterium tumefaciens.

The particle bombardment method (Klein, et al., (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton flying disc (Bio-Rad Labs). The particles are thenaccelerated into the maize tissue with a Biolistic PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovers a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm, et al., (1990) Bio/Technology 8:833-839).

Example 6

This example describes expression of transgenes in dicot cells.

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle,et al., (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μmgold particle suspension is added (in order):5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M).The particle preparation is then agitated for three minutes, spun in amicrofuge for 10 seconds and the supernatant removed. The DNA-coatedparticles are then washed once in 400 μL 70% ethanol and resuspended in40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicatedthree times for one second each. Five microliters of the DNA-coated goldparticles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7

This example describes expression of a transgene in microbial cells.

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg, et al., (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier, et al., (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°C. Cells are then harvested by centrifugation and re-suspended in 50 μLof 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One microgram of proteinfrom the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

Example 8 Isolation of the CesA10, 11 and 12 Genes and their Relevanceto Cell Wall Synthesis and Stalk Strength in Maize

All three genes were isolated from a library made from the zone of anelongating corn stalk internode between the elongation zone and the mostmature part of the internode, the “transition zone”. The library wasmade, subtracted, and sequenced as described in the preceding Examples1, 2 and 3. A genomic database search was conducted as described inExample 4. Derived polypeptide sequences of all the Expressed TagSequences (ESTs) showing homology to the 1 kb 5′-end of any of the 9previously known ZmCesA genes were aligned with the protein sequences ofthe latter. The sequences that did not fully match any of the knowngenes were sequenced from both ends of the respective cDNA clones. Threenew, full-length genes, ZmCesA10 (SEQ ID NO: 25), ZmCesA11 (SEQ ID NO:27) and ZmCesA12 (SEQ ID NO: 29) were isolated by this method.

The polypeptide sequences of the three genes derived from the cDNAsequences (SEQ ID NOS: 26, 28 and 30, respectively) clustered with theCesA genes from other species where they are known to be involved insecondary wall formation (FIG. 4). AtCesA7 and AtCesA8 have been foundto make secondary wall in the vascular bundles (Taylor, et al., (2000).Multiple cellulose synthase catalytic subunits are required forcellulose synthesis in Arabidopsis. Plant Cell, (2000) 12:2529-2539.).Retrotransposon insertions into OsCesA4 and OsCesA7 resulted in abrittle culm phenotype in rice (Katsuyuki Tanaka, Akio Miyao, KazumasaMurata, Katsura Onosato, Naoko Kojima, Yumiko Yamashita, Mayuko Harada,Takuji Sasaki, Hirohiko Hirochika, 2002, Analysis of rice brittlemutants caused by disruption of cellulose synthase genes OsCesA4 andOsCesA11 with the retrotransposon tos17. Plant, Animal & Microbe GenomesX. San Diego, Calif. Abs. Number 324). Each of the genes, ZmCesA 10, 11or 12, groups with one or the other CesA gene from Arabidopsis or riceknown to be involved in secondary wall formation and thus in determiningtissue strength (FIG. 4). The CesA genes derived from the tissuesspecializing in secondary wall formation from other species (Gossypium,Zinnia, Populus) also group into the same clades with the aforementionedgenes.

Further evidence that the maize genes are involved in secondary wallformation and thus in determining stalk strength was obtained from theirexpression pattern using the Massively Parallel Signature Sequencing(MPSS) technology (Brenner, et al., (2000), In vitro cloning of complexmixtures of DNA on microbeads: Physical separation of differentiallyexpressed cDNAs.Proceedings-of-the-National-Academy-of-Sciences-of-the-United-States-of-America(Feb. 15, 2000) 97:1665-1670.; see also, Brenner, et al., (2000) Geneexpression analysis by massively parallel signature sequencing (MPSS) onmicrobead arrays. Nature-Biotechnolog, [print] (June 2000) 18:630-634;see also, Dhugga, (2001), Building the wall: genes and enzyme complexesfor polysaccharide synthases, Curr. Opin. Plant Biol. 4:488-493). Allthree genes are expressed in the tissues rich in cell wall content,supporting their involvement in secondary wall formation as deduced fromtheir relationship to the genes from the other, aforementioned speciesknow to play this role (FIG. 5). All three genes are expressed nearlyidentically across multiple tissues as seen from the correlationcoefficient matrix (Table 3), further strengthening the argument thatthey are involved in secondary wall formation in the vascular bundlesand thus in determining tissue strength.

Correlation among the expression level of the different CesA genes frommaize as studied from Lynx are shown in Table 3.

TABLE 3 CesA1 CesA2 CesA3 CesA4 CesA5 CesA6 CesA7 CesA8 CesA10 CesA11CesA12 CesA1 1 CesA2 0.59 1.00 CesA3 0.07 −0.15 1.00 CesA4 0.44 0.55−0.10 1.00 CesA5 −0.20 −0.29 0.45 −0.33 1.00 CesA6 0.56 0.14 0.14 0.08−0.13 1.00 CesA7 0.68 0.76 −0.06 0.57 −0.29 0.32 1.00 CesA8 0.59 0.73−0.16 0.58 −0.36 0.26 0.61 1.00 CesA10 0.27 0.37 −0.27 0.33 −0.26 0.020.33 0.36 1.00 CesA11 0.39 0.47 −0.22 0.38 −0.28 0.11 0.40 0.42 0.951.00 CesA12 0.34 0.49 −0.27 0.37 −0.31 0.08 0.44 0.45 0.95 0.95 1.00

The correlation matrix was derived from the expression, measured in PPM,from 65 different tissue libraries. Note the nearly perfect correlationamong the expression pattern of the CesA10, 11 and 12 genes.

Example 9

This example describes a procedure to identify plants containing Muinserted into genes of interest and a strategy to identify the functionof those genes. This procedure was also described in U.S. patentapplication Ser. No. 09/371,383 which disclosed members of the same genefamily as the present application. One of skill in the art could readilyconceive of use of this procedure with the any of the Cellulose Synthase(CesA) sequences disclosed in the current application. The currentexample is based on work with the CesA11 gene, identified as SEQ ID NO:27 herein.

The Trait Utility System for Corn (TUSC) is a method that employsgenetic and molecular techniques to facilitate the study of genefunction in maize. Studying gene function implies that the gene'ssequence is already known, thus the method works in reverse: fromsequence to phenotype. This kind of application is referred to as“reverse genetics”, which contrasts with “forward” methods that aredesigned to identify and isolate the gene(s) responsible for aparticular trait (phenotype).

Pioneer Hi-Bred International, Inc., has a proprietary collection ofmaize genomic DNA from approximately 42,000 individual F₁ plants(Reverse genetics for maize, Meeley and Briggs, (1995) Maize Genet.Coop. Newslett. 69:67-82). The genome of each of these individualscontains multiple copies of the transposable element family, Mutator(Mu). The Mu family is highly mutagenic; in the presence of the activeelement Mu-DR, these elements transpose throughout the genome, insertinginto genic regions, and often disrupting gene function. By collectinggenomic DNA from a large number (42,000) of individuals, Pioneer hasassembled a library of the mutagenized maize genome.

Mu insertion events are predominantly heterozygous; given the recessivenature of most insertional mutations, the F₁ plants appear wild-type.Each of the F₁ plants is selfed to produce F₂ seed, which is collected.In generating the F₂ progeny, insertional mutations segregate in aMendelian fashion so are useful for investigating a mutant allele'seffect on the phenotype. The TUSC system has been successfully used by anumber of laboratories to identify the function of a variety of genes(Cloning and characterization of the maize An1 gene, Bensen, et al.,(1995) Plant Cell 7:75-84; Diversification of C-function activity inmaize flower development, Mena, et al., (1996) Science 274:1537-1540;Analysis of a chemical plant defense mechanism in grasses, Frey, et al.,(1997) Science 277:696-699; The control of maize spikelet meristem fateby the APETALA2-like gene Indeterminate spikelet 1, Chuck, et al.,(1998) Genes and Development 12:1145-1154; A SecY homologue is requiredfor the elaboration of the chloroplast thylakoid membrane and for normalchloroplast gene expression, Roy and Barkan, (1998) J. Cell Biol.141:1-11).

PCR Screening for Mu insertions in CesA11:

Two primers were designed from within the CesA11 cDNA and designated asgene-specific primers (GSPs):

Forward primer (GSP1/SEQ ID NO. 32): 5′- TACGATGAGTACGAGAGGTCCATGCTCA-3′ Reverse primer (GSP2/SEQ ID NO. 33): 5′- GGCAAAAGCCCAGATGCGAGATAGAC-3′ Mu TIR primer (SEQ ID NO. 34): 5′- AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC-3′

Pickoligo was used to select primers for PCR. This program chooses theTm according to the following equation:Tm=[((GC*3+AT*2)*37-562)/length]−5

PCR reactions were run with an annealing temperature of 62° C. and athermocycling profile as follows:

Gel electrophoresis of the PCR products confirmed that there was nofalse priming in single primer reactions and that only one fragment wasamplified in paired GSP reactions.

The genomic DNA from 42,000 plants, combined into pools of 48 plantseach, was subjected to PCR with either GSP1 or GSP2 and Mu TIR. Thepools that were confirmed to be positive by dot-blot hybridization usingCesA11 cDNA as a probe were subjected to gel-blot analysis in order todetermine the size of fragments amplified. The pools in which cleanfragments were identified were subjected to further analysis to identifythe individual plants within those pools that contained Mu insertion(s).

Seed from F₁ plants identified in this manner was planted in the field.Leaf discs from twenty plants in each F₂ row were collected and genomicDNA was isolated. The same twenty plants were selfed and the F₃ seedsaved. Pooled DNA (from 20 plants) from each of twelve rows wassubjected to PCR using GSP1 or GSP2 and Mu TIR primer as mentionedabove. Three pools identified to contain Mu insertions were subjected toindividual plant analysis and homozygotes identified. The Mu insertionsites with the surrounding signature sequences are identified below:

Allele 1: 5′-TGGCGGCCG(SEQ ID NO: 35)-Mu-TCTGAAATG (SEQ ID NO: 36)-3′Allele 2: 5′-GCCCACAAG(SEQ ID NO: 37)-Mu-CATCCTGGT (SEQ ID NO: 38)-3′Allele 3: 5′-GTGTTCTTC(SEQ ID NO: 39)-Mu-GCCATGTGG (SEQ ID NO: 40)-3′

All three insertions are within 500 nucleotides of each other in theopen reading frame, suggesting that this region in the gene mightrepresent a hot spot for Mu insertion. One of the insertions, allele 1,is in the region upstream of the predicted six transmembrane domainsnear the C-terminal end of the protein. Each of these insertions isexpected to inactivate the gene since they are all in the exonic regionsof the gene.

Example 10

This example describes the method used to measure mechanical strength ofthe maize stalks as well as the effect of the overexpression ofdifferent CesA genes on stalk strength. The mechanical strength of themature corn stalks was measured with an electromechanical test system.The internodes below the ear were subjected to a 3-point bend test usingan Instron, model 4411 (Instron Corporation, 100 Royall Street, Canton,Mass. 02021), with a span-width of 200 mm between the anchoring pointsand a speed of 200 mm/min of the 3^(rd) point attached to a load cell.For measuring rind puncture strength, a needle was mounted on the loadcell of the Instron and the load taken to puncture the rind was used asa measure of rind puncture strength.

Load needed to break the internode was used as a measure of mechanicalstrength. The internodes are stronger toward the base of the stalk. Thismechanical stalk breaking strength or the “load to break” was used toclassify the hybrids with known stalk characteristics into respectivecategories based on the internodal breaking strength. The load to breakthe internodal zone was very similar to the lodging score that had beenassigned to the hybrids based on field observations (see, FIG. 1).Approximately 90% of the variation for internodal breaking strength wasexplained by unit stalk dry matter below the ear (47%), stalk diameter(30%) and rind puncture resistance (10%). Moisture levels above 30% inthe stalk tissue masked the contribution of the rind tissue to breakingstrength. The internodal breaking strength was highly correlated withthe amount of cellulose per unit length of the stalk.

Four of the CesA genes were expressed under the control of a weakconstitutive promoter, F3.7 (see, Coughlin, et al., U.S. patentapplication Ser. No. 09/387,720, filed Aug. 30, 1999). Table 4 disclosesthe construct numbers, corresponding sequence IDs from the patent,promoters, and the gene names. In2 is an inducible promoter from the In2gene from maize. The In2 promoter responds to benzenesulfonamideherbicide safeners (see, Hershey, et al., (1991) Mol. Gen. Genetics227:229-237 and Gatz, et al., (1994) Mol. Gen. Genetics 243:32-38).

TABLE 4 Construct CesA SEQ ID NO. Promoter Gene name 1 1 F3.7 CesA1 2 9F3.7 CesA4 3 13 F3.7 CesA5 4 17 F3.7 CesA8 5 Control IN2 GUSINT

Twenty-five individual T₀ events for each construct were generated in ahybrid maize background using Agrobacterium-mediated transformation.Data for various traits, such as plant height, stalk mass below ear,stalk diameter, internodal breaking strength and structural material andcellulose percentages in the internodal tissue were collected.

The plants from the transgenic events generated using the CesA8 genewere significantly taller in comparison to the control plants containinga GUS gene. Interestingly, a reduction in height was observed when theCesA1 gene was introduced. The other two genes, CesA4 and CesA5, did notdiffer from the control plants. (See, FIG. 6.) It has long been knownthat cellulose synthase occurs as a terminal rosette complex consistingof multiple functional cellulose synthase polypeptides that areorganized in a ring with a hexagonal symmetry. Each of the six membersof the ring is believed to contain six or more functional enzyme units.In general, 36 or more cellulose chains are extruded simultaneous totheir synthesis through the plasma membrane into the apoplast. Thesechains are crystallized into a microfibril right as they come in contactwith each other after extrusion through the rosette complex. Afunctional cellulose synthase is believed to consist of two polypeptidesderived form different CesA genes, forming a heterodimer, resulting in atotal of 72 or more CesA polypeptides in each rosette.

While not intending to be limited to a single theory, it is possiblethat a homodimer could also form a functional enzyme. Therefore, thepossible reasons for a reduction in plant height in the events whereCesA1 was overexpressed are: 1) the other CesA gene with which itspolypeptide forms a heterodimer is down-regulated and 2) the expressionof the other gene is not affected but the CESA1 homodimer forms anonfunctional enzyme, in which case the functional dimers are competedout of the rosette complex. In the latter case, the overexpressed genebehaves as a dominant repressor of cellulose synthesis. This shouldmanifest in the form of microfibrils with fewer cellulose chains. Thiscould be detected by some physical techniques such as differentialscanning calorimetry (DSC). The reverse could be true for the CesA8 genewhose homodimers may be functional, and/or whose overexpression mightinduce the expression of its partner gene the product of which it usesto make a functional enzyme. The fact that an increase in height isobserved may result from stalk becoming an active sink when CesA8 isoverexpressed. Stalk is usually considered to be a passive sink whichcannot compete well with the developing ear. This argument is supportedby the observation that the plants containing CesA8 as a transgene hadsmaller ears.

Cellulose content and stalk length below the ear is highly correlatedwith the breaking strength of the stalk (see, FIG. 3). An increase incellulose production can be accommodated by the followingalterations: 1) synthesis of the other cell wall constituents staysconstant, leading to an increased cellulose percentage in the wall and2) increase in cellulose synthesis upregulates the synthesis of theother cell wall constituents as well, in which case the percentage ofcellulose does not change in the wall but the amount of cellulose in aunit length does. Two of the CesA genes, CesA4 and CesA8, showed anincrease in the amount of cellulose in a unit length of the stalk belowthe ear (see, FIG. 7). One of the genes, CesA5, did not have any effecton the amount of cellulose in the stalk. It was recently suggested,based on its expression pattern in different tissues, that CesA5 mightactually be involved in the formation of some non-cellulosicpolysaccharide, most probably mixed-linked glucan (Dhugga, (2001) Curr.Opin. Plant Biol. 4:488-493). The data in the accompanying figure seemto support this argument.

The internodes were subjected to breakage with a 3-point Instron and theload to break plotted as a function of unit cellulose amount. (See, FIG.2.) A high correlation between these two traits is observed from themultiple events, particularly for CesA8 (FIG. 3). We have found fromother studies that this gene is involved in cellulose synthesis in thevascular bundles in the elongating cells (Holland, et al., (2000) PlantPhysiol. 123:1313-1323). These data support our previous observationsand supports the observation that the amount of cellulose in a unitlength of stalk below the ear results in an increased stalk strength.

Example 11

This example describes the method used to overexpress CesA genes whichwill increase the quality of harvested stover, leading to an increase inethanol yield per unit stover.

Transgenic plants expressing the Ces A gene of interest could beproduced by the method outlined in Example 5 or other suitable methods.These plants containing increased quantities of cellulose would then beused to produce higher quality stover.

The following is an example of the applications of the present inventionin applications of ethanol biorefineries. In addition the cellulosebiosynthetic pathway's role as primary determinant of tissue strength, atrait that is of significant interest in agriculture, where celluloseconstitutes the most abundant renewable energy resource. More than 200million metric tons of stover is produced just from maize in the UnitedStates every year. About one-third of this could potentially be utilizedin ethanol biorefineries (Kadam and McMillan, 2003). The worldwideproduction of lignocellulosic wastes from cereal stover and straw isestimated to be ˜3 billion tons per year (Kuhad and Singh, 1993). Stovermaterial containing higher amounts of cellulose and lower amounts oflignin is expected to increase ethanol production in the biorefineries.Lignin is a target for reduction because it is an undesirableconstituent in paper industry as well as in silage digestibility (Hu, etal., 1999; Li, et al., 2003).

Corn stover alone offers a significant target as a feedstock for theethanol biorefineries (see, the World Wide Web atctic.purdue.edu/Core4/ctic-dc.ppt; andbioproducts-bioenergy.gov/pdfs/bcota/abstracts/31/z 263.pdf.) Aside fromits use in biorefineries, it can substitute hardwood fiber for paperproduction. With rapid progress being made in streamlining the processof fermentation of stover material and increasing cost of imported oil,corn stover is expected to become a key feedstock in ethanol and paperproduction (Wheals, et al., 1999; Atistidou and Penttila, 2000). Inaddition to supplying 5-8 billion gallons of ethanol per year with noadditional land use, it is expected to contribute to an annual farmincome of $2.3 billion and reduce the greenhouse gases by 60-95 millionmetric tons, which is 12-20% of the US-Kyoto commitment (see, the WorldWide Web at ctic.purdue.edu/Core4/ctic-dc.ppt; andbioproducts—bioenergy.gov/pdfs/bcota/abstracts/31/z 263.pdf). Ethanolcombustion results in carbon dioxide and water, the same molecules plantprimarily uses to make biomass.

The concern about there being an effect on the soil organic matter bythe removal of the aboveground biomass is mitigated by the findings thatover a 30-year period, no significant difference was observed in thesoil organic matter between a field where the aboveground stover wasremoved for silage and the one where the stover was ploughed into theground after grain harvest. Most of the ploughed stover is lost ascarbon dioxide into the atmosphere.

During pretreatment of the corn stover for enzymatic digestion, thesoluble sugars are discarded. Also, pentose sugars are not as wellfermented as the hexose sugars despite the progress made in thefermentation process, which involves using Zymomonas bacteria instead ofthe traditional yeast (Atistidou and Penttila, 2000; Badger, 2002). Thepolysaccharide fraction of the corn stalk contains ˜20% pentose sugars,the remainder being hexose sugars (Dhugga, unpublished). Also, the freesugar concentration ranges from 4-12%. Lignin content averages ˜19% andranges from 18-23%. By overexpressing the CesA genes of the presentinvention, the free sugars can be converted into polymeric (cellulosic)form, which will increase ethanol yield per unit of the harvestedstover. The claimed invention also teaches an increase cellulose at theexpense of pentose-containing polymers (e.g., arabinoxylan) and lignin.

Example 12

This example discusses the application of CesA genes in late seasonstalk strength.

Stalk lodging results in significant yield losses in crop plants,particularly in cereals (Duvick and Cassman, 1999). Stalk standabilityis dependent upon the amount of dry matter per unit length of the stalkand is thus a function of resource partitioning and allocation. Harvestindex, the ratio of the grain to total aboveground biomass, is anindicator of dry matter partitioning efficiency. It has remained around50% for over a hundred years in maize (Sinclair, 1998). In comparison tomaize, harvest index acquired a different role in increasing plantstandability in small grain cereals where it was significantly increasedwith the introduction of dwarfing genes. Reduced stature made thesecereals less likely to lodge by reducing torque on the top-heavy straw,which allowed for higher inputs such as fertilizers and irrigation,resulting in increased biomass production per unit land area. Whereasyield increases in small grain cereals have resulted from an increase inboth harvest index and total biomass production per unit land area,those in maize have been the consequence of mainly an increase in totalbiomass. Increased planting density as a means of increasing grain yieldin maize has affected changes in leaf angle and shape as adaptations tothis environment and has in general resulted in increased plant and earheights (Duvick and Cassman, 1999). The stalk becomes mechanicallyweaker with increasing planting density because of reduction inindividual plant vigor that results from a nonlinear relationshipbetween planting density and biomass increase.

The understanding that cellulose in a unit length of the stalk is indeedthe main determinant of mechanical strength had been proposed.(Appenzeller, et al., 2004). Most of the dry matter and thus cellulosein the stalk is concentrated in the outer layers, collectively referredto as rind, which is composed of densely packed vascular bundles.Vascular bundles are surrounded by sclerenchymatous cells. Althoughvascular bundles are also sparsely distributed in the internal tissue, agreat majority of them are present in the outer layers as judged fromthe dry matter distribution (FIG. 8). FIG. 8 describes the contributionof different stalk components to dry matter, diameter, volume and stalkstrength in maize hybrids. The data are derived from seven hybrids grownat three densities (27, 43 and 59 K per acre) in three replications eachin 2001. Two stalks were sampled from each replication. Internodes 3 and4 below the ear were broken with Instron. After breaking, the 3rdinternode was separated into rind and inner tissue. Path coefficientanalyses were performed using rind and inner tissue as independentvariables (X₁ and X₂, respectively) and the whole stalk as the dependentvariable (Y). The multiple regression equation: Y=a+b₁X₁+b₂X₂+e where ais the intercept and e error. Path coefficients were calculated asfollows: ρYXn=b_(n)*δ_(n)/δ_(Y) where n is 1 or 2. The contribution ofeach independent variable to whole stalk (Y) was calculated as follows:ρYxn*rYxn where r is the correlation coefficient. Note: the unexplainedvariation for diameter is attributable to the corn stalk not beingperfectly round and the difficultly thus associated with determining thecross-sectional area accurately. Some other variable, like size andnumber of vascular bundles and their density, may account for theremaining variation in strength. The introduced transgene, by removingthe limitation of the particular step it encodes the enzyme forcatalyzing, may either lead to an increase in the percentage of thatparticular polysaccharide (composition changed) or of the whole cellwall (composition not changed). In the latter case, the additional drymatter could be accommodated in enlarged vascular bundles which could,in turn, result in an increased diameter.

Isolation of genes that affect cellulose formation has made it possibleto test their respective roles in stalk strength by transgenic andreverse genetics approaches (Appenzeller, et al., 2004). Transgenicplants expressing the Ces A gene of interest are produced by the methodoutlined in Example 5 or other suitable methods. Expression of thecellulose synthase gene is measured Three of the twelve cellulosesynthase genes are preferentially expressed in the secondarywall-forming cells (Appenzeller, et al., 2004). Whereas two of thesegenes, CesA10 and CesA11, are expressed more highly in the vascularbundles, CesA12 appears to be more highly expressed in the surrounding,sclerenchymatous cells (Appenzeller, et al., 2004). Overexpression ofthe three CesA genes individually and in combinations is used toincrease cellulose production in the rind cells as well as the internaltissue cells. Internal tissue cells, as shown in FIG. 8, account for amajority of the volume but only a small amount of biomass and thus offersuitable targets for making more cellulose. Isolated promoters for eachof these genes are used to drive the expression of these genes indifferent cell types. In addition, promoters from other genes can beused to express the CesA genes in other cell types.

Example 13

This example discusses the application of the CesA genes in improvingnodal strength to reduce mid-season green snap.

Mid-season green snap is a significant problem in the Western plains,e.g., Nebraska, North and South Dakota and Western Minnesota, wherebythe stalk snaps at the nodal plate before flowering in a severewindstorm at or below the ear node, resulting in yield losses of up to80%. The underlying reason for this lesion is the disparity in the ratesof elongation growth and dry matter deposition in the corn plant beforeflowering. Whereas a plant doubles in height in approximately two weeksbefore flowering, the most rapid rate of elongation growth, itaccumulates only 30% additional dry matter during the same period,resulting in a 3-fold disparity (Dhugga, unpublished data). The plantthus becomes susceptible to breakage. It has been determined that thebreakage occurs through the pulvinal zone at the base of the leafsheath.

Transgenic plants expressing the Ces A genes are produced by the methodoutlined in Example 5 or other suitable methods. The expression patternof eleven of the twelve maize CesA genes in the pulvinal zone tissue isshown in FIG. 9. The twelfth, CesA9, had the same tag at CesA4 to whichit is very highly related. Seven of the genes, CesA1, 4, 7, 8, 10, 11and 12 are expressed at a higher level than the remaining genes. CesA8shows the highest expression in this tissue. The expression of thevarious CesA genes, particularly CesA8, in this tissue can be used toincrease its strength.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, patent applications, andcomputer programs cited herein are hereby incorporated by reference.

1. An isolated cellulose synthase product obtained from a transformedplant tissue expressing an isolated polynucleotide that encodes afunctional cellulose synthase polypeptide having at least 95% identityto SEQ ID NO:
 50. 2. The cellulose synthase product according to claim1, wherein the transformed plant cell is a monocot cell.
 3. Thecellulose synthase product of claim 1, wherein the plant cell isselected from the group consisting of: maize, soybean, sunflower,sorghum, canola, wheat, alfalfa, cotton, rice, barley and millet.
 4. Thecellulose synthase product according to claim 1, which improves stalkstrength of a plant by overexpression of the polynucleotide.
 5. Thecellulose synthase product according to claim 1, which reduces greensnap by improving nodal strength.
 6. The cellulose synthase productaccording to claim 1, which is a constituent of ethanol.
 7. Thecellulose synthase product of claim 1, wherein the polynucleotide of SEQID NO: 49 encodes the functional cellulose synthase polypeptidecomprising SEQ ID NO: 50.